We thank Anne de Jong and Siger Holsappel for help with the DNA microarrays slide production. Anne de Jong&#8217;s support for bioinformatics analysis is also appreciated. We also thank Jelle Slager for reviewing the paper. Muhammad Afzal and Irfan Manzoor are supported by the GC University, Faisalabad, Pakistan under the faculty development program of HEC Pakistan.

1. Preparation of Media, and Cell Culture
<ol>
	<li>Grow S. pneumoniae D39 wild-type strain21 as described previously11. Inoculate cells stored at -80&#160;oC in 10% glycerol (with 1/100 ratio in 50 ml sterile tubes) in 50 ml Chemically Defined Medium (CDM) with a final pH of 6.4 15, but omit L-serine from the amino acid mixture. 
	Note: two different CDMs were used; one containing a minimum concentration of L-serine (150 &#181;M) and the other containing a maximum concentration of L-serine (10 mM). This minimum concentration is basically the concentration of L-serine in human blood plasma and the aim is to compare the gene expression of S. pneumoniae D39 strain under these two conditions.</li>
</ol>
2. Isolation of Total RNA
<ol>
	<li>Materials
	<ol>
		<li>Use acid phenol, RNA grade for the isolation of total RNA.</li>
		<li>Macaloid:
		<ol>
			<li>Suspend 2 grams of macaloid in 100 ml TE and boil for 5 min. Cool to RT and sonicate until macaloid gels.Centrifuge the solution at 2,000 &#215;&#160;g for 5 min&#160;at RT, resuspend in 50 ml TE pH 8 and store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Solutions:
		<ol>
			<li>Treat all solutions with diethyl pyrocarbonate (DEPC). Add 100 &#181;l DEPC/100 ml solution, incubate O/N at 37 &#176;C and autoclave for 15 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Methodology
	<ol>
		<li>Grow 50 ml of S. pneumoniae D39 cell culture in 50 ml tubes at 37 oC (no shaking) until mid-exponential phase (OD ~0.3). Centrifuge cultures at 4 &#176;C on 10,000 &#215;&#160;g&#160;for 2 min. Discard media and immediately freeze the cell pellets in liquid nitrogen.</li>
		<li>Premix 300 &#181;l chloroform:IAA (24:1) and 300 &#181;l phenol (acid phenol, RNA grade). Use 500 &#181;l of the organic phase in step 2.2.3.</li>
		<li>Prepare the following mixture in advance in RNA-free screw-cap tubes: 0.5 g glass beads, 50 &#181;l 10% SDS, 500 &#181;l phenol/chloroform:IAA (as prepared in step 2.2.2), macaloid layer (150-175 &#181;l, not exact as it is very viscous). Resuspend the cell pellets in 400 &#181;l TE (DEPC) and add the resuspended cells into the screw-cap tubes.</li>
		<li>To break the cells, place the screw-cap tubes in a bead beater for 2x 60&#8221; pulses (&#8216;homogenize&#8217;) with 1 min&#160;interval on ice. Centrifuge the samples for 10 min&#160;at 10,000 &#215;&#160;g&#160;(4 &#176;C).</li>
		<li>Transfer the upper phase to a fresh tube, add 500 &#181;l chloroform:IAA (24:1) and centrifuge for 5 min at 10,000 &#215;&#160;g (4 &#176;C).</li>
		<li>Transfer 500 &#181;l of the upper phase to fresh tubes, add 2 volumes (1 ml) of lysis/binding buffer and mix by pipetting up and down. Isolate total RNA using the RNA isolation kit and follow the manufacturer recommended protocol.</li>
	</ol>
	</li>
</ol>
3. RNA&#160;Cleanup
<ol>
	<li>To remove the contamination of DNA from total RNA, add 100 &#181;l DNAseI mix (90 &#181;l DNAse buffer and 10&#181;l DNAseI) and incubate for 20-30 min at 15-25 &#186;C.</li>
	<li>Wash cleaned RNA using the RNAse kit. Obtain 50 &#181;l of eluted volume.</li>
</ol>
4. Analysis of RNA
<ol>
	<li>Determine the concentration of RNA on a spectrophotometer. Determine the quality of RNA by using an assay as follows (Figure 2).
	<ol>
		<li>Dilute 1 &#181;l of sample with 50 &#181;l of DEPC water to get a concentration of 20-200 ng/&#181;l.</li>
		<li>Use 1 &#181;l diluted RNA sample to check quality on the Bioanalyser according to the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Note: a ratio of 23S:16S of around 2.0 is considered good.1 A260 unit RNA corresponds to 40 &#181;g/ml. A recommended amount for labeling is 10-20 &#181;g.</li>
</ol>
5. cDNA Preparation and Labelling
NOTE: The following protocol was followed for cDNA preparation and labelling.
<ol>
	<li>Annealing
	<ol>
		<li>Perform annealing reaction in 300 &#181;l PCR tubes, keeping the concentration of total RNA 10-15 &#181;g for homemade slides or 5 &#181;g for company-made slides. Mix RNA with 2 &#181;l Random nonamers (1.6 &#181;g/&#181;l) and add nuclease-free water if necessary to keep the final volume of the annealing mixture to 18 &#181;l.</li>
		<li>Keep the annealing mixture at 70 &#176;C for 5 min. After that, cool down the mixture to RT&#160;for 10 min (annealing) and if needed, spin down the reactions to the bottom of the tube. Place the reaction tubes on ice for at least 1 min.</li>
	</ol>
	</li>
	<li>Reverse Transcription
	<ol>
		<li>Prepare the reverse transcriptase mix as follows: to 18 &#181;l annealing mix, add 12 &#181;l Master mix (consisting of 6 &#181;l 5x First Strand buffer, 3 &#181;l 0.1 M DDT, 1.2 &#181;l 25x AA-dUTP / nucleotide mix and 1.8 &#181;l reverse transcriptase (1 &#181;l for company-made slides). Keep the reaction mix for 2-16 hr at 42 &#176;C.</li>
	</ol>
	</li>
</ol>
6. Degradation of mRNA and Purification of cDNA
<ol>
	<li>To degrade the mRNA from the reaction mixture, add 3 &#181;l of 2.5 M NaOH and place at 37 &#176;C for about 15 min. Subsequently, add 15 &#181;l of 2M HEPES free acid to neutralize the NaOH.</li>
	<li>Purify the cDNA mixture by using PCR clean-up columns and follow the manufacturer&#8217;s protocol.</li>
</ol>
7. Measurement of cDNA Concentration
<ol>
	<li>Measure cDNA concentrations on a spectrophotometer. To continue with labelling, check that the concentration of cDNA is at least 60 ng/&#181;l (homemade slides) or 20 ng/&#181;l (company-made slides).</li>
</ol>
8. Labelling of cDNA with Amine-reactive Dye and Purification
<ol>
	<li>Use amine-reactive dye to label the cDNA. Directly mix the cDNA with one aliquot (5 &#181;l) of amine-reactive dye.</li>
	<li>Incubate the mixture at RT, in the dark, for 60 to 90 min and proceed directly to purification of dye-labelled cDNA. Purify dye-labelled cDNA by using PCR clean-up columns and following the manufacturer&#8217;s protocol. Elute cDNA in 50 &#181;l of elution buffer.</li>
</ol>
9. Measurement of Labelled cDNA
<ol>
	<li>Use a spectrophotometer to measure the incorporation of amine-reactive dyes into the cDNA. Check that the concentration of amine-reactive dyes is at least 0.5 pmol/&#181;l in a total volume of 50 &#181;l.</li>
</ol>
10. Mixing of Labelled cDNA Samples
<ol>
	<li>For homemade slides, use all the labelled cDNA for hybridization with not more than 30% difference in cDNA concentration. For company-made slides, use cDNA with not more than 2-fold difference in cDNA concentration. 
	Note: normally, about 300 ng of cDNA is needed for company-made slides, which is very little as compared to the required amount of cDNA for homemade slides.</li>
</ol>
11. Hybridization and Washing
<ol>
	<li>Use the following reagents: demi water, ethanol 99%, SHY Buffer (homemade; with&#160;40 &#181;l yeast RNA). Prepare maximum 1 ml of SHY.</li>
	<li>Apparatus and solutions preparation
	<ol>
		<li>Switch on vacuum concentrators and heater at least 1 hr before drying. Switch on hybridization oven, with set point adjusted to the correct hybridization temperature (for S. pneumoniae DNA microarrays, use 45 &#176;C). Similarly, preheat hybridization cassette and oven for 30-60 min.</li>
		<li>Preheat SHY buffer at 68 &#730;C for at least 30 min.</li>
	</ol>
	</li>
	<li>Sample preparation (labelled cDNA)
	<ol>
		<li>Combine equal quantities of labelled cDNAs (max 30% difference). Dry the sample using the vacuum concentrators at high temp (approx. 40 min) until the volume is smaller than 7 &#181;l.</li>
	</ol>
	</li>
	<li>Lifter-slip
	<ol>
		<li>Use clean lifter-slips. Note: &#177; 30 &#181;l can be loaded on the slide.</li>
		<li>Clean the lifter-slips with soap, plenty of tap water and 100% ethanol. Note: Dirty lifter slips give high background.</li>
		<li>Air dry the lifter-slips with air pistol to blow away dust particles. Place a clean lifter-slip on the slide with the white teflon lining at the sides facing down.</li>
	</ol>
	</li>
	<li>Adding hybridization buffer (to labelled cDNA)
	<ol>
		<li>Dissolve the dried dye samples in 7 &#181;l H2O and incubate at 94 &#176;C for 2 min.</li>
		<li>Immediately, add 35 &#181;l preheated SHY buffer (68 &#176;C), mix gently and spin at maximum speed for 1 min to get rid of precipitates. Preheat the probe at 68 &#730;C for approximately 5 min until loading.</li>
	</ol>
	</li>
	<li>Slide-lifter-slip assembly and preheating.
	<ol>
		<li>Place the hybridization slide holder on a heat-block at 50 &#730;C. Place DNA microarray slides with the lifter-slip on the heated hybridization slide holder and preheat the slide with lifter-slip for a minute. Perform the next steps as quickly as possible.</li>
		<li>Add 40 &#181;l of the sample target to the end of the slide. Allow the fluid to flow between the glass surfaces by capillary force. Perform all pipetting slowly and carefully.</li>
		<li>Keep the slides with lifter-slip horizontal at all times and move slowly to make sure that the lifter-slip did not move.</li>
		<li>Take the pre-warmed hybridization cassette out of the hybridization oven and close the machine. Place filter-paper soaked with 3 ml 2x SSC (standard saline citrate) in the hybridization cassette.</li>
		<li>Gently place the hybridization slide holder with slides in the hybridization cassette. Close the hybridization cassette and put in the hybridization oven again (for about 16-18 hr).</li>
	</ol>
	</li>
	<li>Slide washing
	<ol>
		<li>Prepare fresh wash-buffers I, II and III (750 ml per wash step). For 500 ml wash-buffer I, use 2 x SSC / 0.5 % SDS. For 500 ml wash-buffer II, use 1 x SSC / 0.25 % SDS. For 500 ml wash-buffer III, use 1 x SSC / 0.1 % SDS (optional)</li>
		<li>Place the wash-buffers at 30 &#176;C (to be sure SDS is dissolved).</li>
		<li>Submerge the slides as quickly as possible, but very gently, in a falcon tube filled with 50 ml wash buffer I until the glass rests on the conical bottom of the tube.</li>
		<li>After a few seconds, when the lifter-slip sinks to the bottom of the tube, take out the slide with tweezers without scratching the array and put in the rack of the wash station. Continue with the washing with no time gap.</li>
		<li>Wash the slides for 5 min in 500 ml wash-buffer I (in a washing station). Give it a second wash for 20-30 min in 500 ml wash-buffer II (in a washing station). Similarly, wash the slides for 5 min in 500 ml wash-buffer III (optional) (in a washing station).</li>
		<li>Dry the slides for 2 min at 2,000 rpm.</li>
	</ol>
	</li>
</ol>
12. Microarray Analysis
<ol>
	<li>Scan images with respective wavelengths in scanner and save in a folder &#8220;Jove Project&#8221;.</li>
	<li>Use software to analyze the scanned files initially as described previously 22. After running this software, select the &#8220;Open Image&#8221; tab to load the red image file (-.550) as &#8220;Red&#8221; and green image file (-.635) as &#8220;Green&#8221;. Upload the gal file (.gal) having S. pneumoniae array list on image file by selecting &#8220;Load Array List&#8221; tab 22. 
	Note: this array list consisted of 48 grids, on each grid there were 16 rows and 15 columns. Each spot on the grid represents a single gene and spot information including gene name is added through a spot description file. The spot numbers are given from left to right and top to bottom.</li>
	<li>After carefully spotting the grids, select tab &#8220;Find Array, Find Blocks, Align Features&#8221; to align the spots. After aligning all the features, analyze the image by selecting the &#8220;Analyzing&#8221; tab. Create a new file having results, histogram and scatter plot. Save this file from tab &#8220;Save results as&#8221; as a .gpr file for further analysis.</li>
	<li>Perform further normalization and processing of data with in-house developed Microprep software package as described9.</li>
	<li>Use independent biological replicates for DNA microarray data which are dye-swapped. Perform CyberT implementation of a variant of t-test1 and calculate false discovery rates (FDRs) as described9.</li>
	<li>For differentially expressed genes, take p &#60; 0.001 and FDR &#60; 0.05 as a standard.</li>
	<li>Upload DNA microarray data on NCBI submission page to get a GEO (Gene Expression Omnibus) accession number.</li>
</ol>

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The authors declare that they have no competing financial interests.

We describe a user-friendly protocol that can be applied to perform whole transcriptome analysis of bacteria. The key point about this particular technique is that the condition under which the cells are harvested will vary. After harvesting the cells and RNA isolation, this technique becomes equal for all types of bacterial samples and follows exactly identical steps and therefore, can be applied to any type of bacterial culture. The protocol is very simple and convenient and starts from RNA isolation. Our RNA isolation...

The study of the whole set of mRNA abundance (transcriptome) encoded by the genome of a unicellular organism or a eukaryotic cell at a specific time or under a specific condition, including gene overexpression or knock-out, is called transcriptomics. Transcriptomics allows us to observe to what extent genes are expressed under a particular condition at a time point X and gives us information about how strongly the genes are expressed relative to a reference.
A microarray is a two-dimensional array on a solid substrate (usually a glass slide or silicon thin-film cell) that can be used to assay large quantities of biological material using high-throughput screening, and miniaturized, multiplexed and parallel processing and detection methods. Microarrays come in various types, including DNA-microarrays, protein microarrays, peptide microarrays, tissue microarrays, antibody microarrays, cellular microarrays and others. A DNA microarray is basically an assembly of microscopic DNA spots fixed to a solid surface, usually glass. DNA microarrays are used to measure the expression levels of a gene or a set of genes simultaneously or to genotype multiple regions of a genome2,3. Picomoles (10-12 moles) of a probe are present within each DNA spot that represents a specific DNA sequence, also known as a reporter. The labelled mRNA molecules from the samples are called &#8216;targets&#8217;. Fluorophores are used to measure probe-target hybridization and detection of fluorophore-labelled targets determines the relative abundance of nucleic acid sequences in the target. A microarray experiment can accomplish multiple genetic tests in parallel because an array may contain tens of thousands of probes. The layout of a simple microarray experiment is shown in Figure 1. Recently, it was established in our and other labs that these arrays are reusable, which makes this technique quite cost-effective.
Different RNA isolation and purification techniques have been developed over the years including C-TAB, SDS and GT methods 4&#8211;8. Furthermore, several commercial kits are also available. For gene expression high quality RNA is very important. Therefore, the RNA isolation methods are modified to get a maximum quantity of RNA. Similarly, the steps for cDNA preparation and labelling of cDNA are minimized. Normalization of data after scanning is also performed efficiently by using in-house built software packages and tools9.
Streptococcus pneumoniae is a Gram-positive human pathogen that colonizes the nasopharynx and is the cause of multiple infections such as pneumonia, sepsis, otitis media and meningitis10. The bacterium can utilize a wide variety of the nutrients required for growth and survival 11,12. A number of studies have been carried out on the pneumococcal nitrogen metabolism and regulation emphasizing the importance of amino acids and their role in virulence13,14. In this study, the transcriptomic response of S. pneumoniae to changing concentrations of L-serine, an amino acid abundantly present in the human blood plasma, is reported using DNA microarrays. The transcriptomic response of S. pneumoniae grown in a minimum concentration of L-serine (150 &#181;M) was compared to that grown in a maximum concentration (10 mM) of serine. Chemically defined medium (CDM or minimal medium)15 was used for this study to control the concentration of serine. The focus of this study is to make this technique user-friendly and to provide different tools for data normalization and analysis. Therefore, a number of tools were developed for analysis and data interpretation. FIVA (Functional Information Viewer and Analyzer) provides a platform for processing information contained in clusters of genes having similar gene expression patterns and for constructing functional profiles16. PROSECUTOR is another software package that facilitates the identification of putative functions and annotations of genes 17. By making use of clustering methods, DISCLOSE provides a DNA binding site detection algorithm. Cis-regulatory motifs of genes can be projected by using this algorithm 18. Genome2D offers a Windows-based platform for visualization and analysis of transcriptome data by offering different color ranges to characterize the changes in gene expression levels on a genome map19. The PePPER webserver offers, in addition to the all-in-one analysis method, a toolbox for mining for regulons, promoters and transcription factor binding sites 20. Full annotation of intergenic regions in a bacterial genome can be achieved by using this package. Biologists can greatly benefit from PePPER as it offers them a platform for designing experiments so that the hypothesized information can be confirmed in vitro20. These software packages contribute significantly to the microarray analysis as most of them are freely available and make data normalization and analysis very reliable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acid phenol</td>
			<td>SigmaAldrich</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr>
			<td>Roche RNA isolation kit</td>
			<td>Roche Applied Science</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td></td>
			<td>105015</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Boom</td>
			<td>92013505.1000.</td>
			<td></td>
		</tr>
		<tr>
			<td>IAA</td>
			<td></td>
			<td>106630</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Nanodrop</td>
			<td>ND-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent BioAnalyser</td>
			<td>Agilent</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>AA-dUTP</td>
			<td>Life technologies Invitrogen</td>
			<td>AM8439</td>
			<td></td>
		</tr>
		<tr>
			<td>DDT</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>First Strand buffer</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SigmaAldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight-550</td>
			<td>Thermoscientiffic</td>
			<td>62262</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight-650</td>
			<td>Thermoscientiffic</td>
			<td>62265</td>
			<td></td>
		</tr>
		<tr>
			<td>SHY Buffer</td>
			<td>SigmaAldrich</td>
			<td>H7033-125ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedvac cooler</td>
			<td>Eppendorf</td>
			<td>RUGNE3140</td>
			<td>Speedvac concentrator plus</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Grant Boekel</td>
			<td>Iso-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Lifter-slips</td>
			<td>Erie Scientific</td>
			<td>25x60I-M-5439</td>
			<td></td>
		</tr>
		<tr>
			<td>Wipe</td>
			<td>KIMTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>SigmaAldrich</td>
			<td>L3771-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>SigmaAldrich</td>
			<td>W302600-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Genpix autoloader 4200A1</td>
			<td>MSD analytical technologies</td>
			<td></td>
			<td>Microarray scanner</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>SigmaAldrich</td>
			<td>104766</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fast and reliable pipeline for bacterial transcriptome analysis case study: serine-dependent gene regulation in streptococcus pneumoniae

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays to study gene expression, but most are limited in terms of providing an overall picture of the expression of the whole transcriptome. DNA microarrays offer a fast and economic research technology, which gives a full overview of global gene expression and have a vast number of applications including identification of novel genes and transcription factor binding sites, characterization of transcriptional activity of the cells and also help in analyzing thousands of genes (in a single experiment). In the present study, the conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis have been optimized. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicabale for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes. Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data.

RNA, cDNA isolations and analysis
L-serine is one of the essential amino acids and its concentration in human blood plasma varies from 60-150 &#181;M in children and adults. Its role in the biosynthesis of purines and pyrimidines highlights its importance in metabolism and it is a precursor to several amino acids (glycine, cysteine and tryptophan). To study the impact of L-serine on the whole transcriptome of S. pneumoniae D39 wild-type strain, microarray analysis of ...

Watch this Scientific Journal Video about A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae at JoVE.com

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages.

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays ...

a-fast-reliable-pipeline-for-bacterial-transcriptome-analysis-case

Research

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10.3K vues.  University of Groningen. This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages. 

Vidéo: A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Bacterial Gene Expression Analysis Using Microarrays

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...