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

The overall goal of this procedure is to provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. This is accomplished by first growing and harvesting bacterial cells under a specific condition. Next, total RNA is isolated from the harvested cells and CDNA is made from the RNA.

Then the CDNAs are labeled and hybridized to in-house made micro assay slides. Finally, the slide is washed after which the slides are scanned and analyzed. Ultimately, the microarray is used to show the transcriptomic changes in bacterial cells under the specific condition.

The main advantage of the DNA microarray technology over other existing methods for studying gene expression like QPCR or northern blotting, is that it provides a global overview of all the transcriptomic changes incurred under a specific condition. It is fast, reliable, and relatively cheap. Demonstrating the procedure will be Mohammed Sal and Airon Manzu, PSD students from my laboratory in molecular genetics After thawing, s pmo, ED 39 cell pellets and Resus suspending in 400 microliters of te according to the text protocol, prepare the following in RNA free screw cap tubes, 0.5 grams of glass beads, 50 microliters of 10%SDS 500 microliters of previously prepared phenol chloroform and the resuspended cell pellets.

To break the cells, place the tubes into a bead beater and homogenize twice for 60 seconds each placing them on ice for one minute in between pulses, then centrifuge the samples at 10, 000 Gs and four degrees Celsius for 10 minutes. Transfer the upper phase to a fresh tube, and at 500 microliters of chloroform i a, a and centrifuge for an additional five minutes. Pipette 500 microliters from the upper phase to fresh tubes.

Add two volumes of lysis binding buffer and mix by pipetting up and and down. Then using an RNA isolation kit isolate total RNA following the manufacturer's protocol to remove contaminating DNA from the RNA. Add 100 microliters of D a's one mix and incubate at 15 to 25 degrees Celsius for 20 to 30 minutes.

Use the RN a's kit to wash the cleaned RNA and Elute into a 50 microliter volume to carry out the an kneeling reaction in 300 microliter PCR tubes. Mix total RNA with two microliters of random monomers and use nuclease free water if necessary to bring the volume up to 18 microliters. Incubate the an kneeling mixture at 70 degrees Celsius for five minutes.

Then cool the reactions down at room temperature for 10 minutes, and give them a quick spin before placing the tubes on ice for at least one minute. To reverse transcribe CDNA, prepare the reverse transcription mix, combine 18 microliters of the mix with 12 microliters of master mix and incubate the reaction at 42 degrees Celsius for two to 16 hours. Degrade the mRNA from the reaction by adding three microliters of 2.5 molar and a OH and place it at 37 degrees Celsius for approximately 15 minutes.

Then add 15 microliters of two molar heaps free acid to neutralize the NAOH. Then after using PCR cleanup columns to purify the CDNA with a spectrophotometer, measure the CDNA concentration to label the CDNA. Add five microliters of an am mean reactive dye to 20 to 60 nanograms per microliter of CDNA.

Place the mixture in the dark and incubated room temperature for 60 to 90 minutes before using PR cleanup columns to purify the dye labeled CDNA eluding in 50 microliters of evolution buffer with a spectrophotometer, ensure that the concentration of labeled CDNA is at least 0.5 pico moles per microliter in a total volume of 50 microliters. After preparing shy buffer, according to the text protocol, preheat the buffer at 68 degrees Celsius for at least 30 minutes. To prepare the sample, combine equal quantities of labeled CDNAs, then use vacuum concentrators at high temperature to reduce the volume to seven microliters or less.

In the meantime, to clean lifter slips to reduce background signal, use soap and plenty of tap water, followed by 100%ethanol after blow drying the lifter slips, place one on a slide with the white Teflon lining facing down. Next, dissolve the dried dye samples in seven microliters of H2O and incubate at 94 degrees Celsius for two minutes. Immediately add 35 microliters of preheated shy buffer, mix gently and spin and maximum speed for one minute.

To pellet precipitates preheat the probe at 68 degrees Celsius for approximately five minutes before loading. To assemble the slide and lifter slip, place the hybridization slide holder on a heat block at 50 degrees Celsius. Place DNA microarray slides with the lifter slip on the heated hybridization slide holder and preheat the slide with the lifter slip for a minute.

Then, as quickly as possible, add 40 microliters of the sample target to the end of the slide. Allow the fluid to flow between the glass surfaces by capillary force. Now remove the prewarm hybridization cassette out of the hybridization oven and close the machine.

Then place filter paper soaked with three milliliters of two XSSC in the hybridization cassette. Gently place the hybridization slide holder with slides in the hybridization cassette. Close the hybridization cassette and place it back in the hybridization oven and incubate for 16 to 18 hours.

For washing the slides, prepare fresh wash buffers one, two, and three, and place them at 30 degrees Celsius. To ensure the SDS dissolves when the incubation is complete, gently submerged the slides as quickly as possible in a Falcon tube filled with 50 milliliters of wash buffer, one until the glass rests at the bottom of the tube. After the lifter slips, sinks to the bottom of the tube without scratching the array, use tweezers to remove the slide and put it in the rack of the wash station.

Immediately use 500 milliliters of wash buffer one to wash the slides for five minutes. Then transfer the slides to wash buffer two for 20 to 30 minutes before transferring them to 500 milliliters of wash buffer three for five minutes. Finally, dry the slides at 2000 RPM for two minutes before carrying out microarray analysis.

According to the text protocol to study the impact of L serin on the whole transcriptome of S pneumonia, D 39 Wildtype strain microarray analysis of the D 39 strain grown in CDM with 150 micromolar of L Seine, compared to that grown in 10 millimolar l Seine in the same medium was performed. This table lists the concentrations of total RNA isolated from each culture as shown in this figure. After removing the DNA, the quality of the RNA was examined.

These two lanes represent RNA from 150 micromolar Seine, and these two lanes are from cultures grown with 10 millimolar seine. The presence of two clear bands indicates good quality RNA. The concentrations of CDNA synthesized from total RNA is recorded in this column and of the AM mean reactive dye labeled samples is listed here.

A scatterplot analysis of the AM mean reactive dies ratio is seen in this figure, and the data was further analyzed to reduce noise. This table summarizes the results of the microarray studies after applying the criteria of greater than or equal to a twofold difference and a P value of less than 0.001. As seen here, a number of genes were differentially expressed in the presence of minimum L serin as compared to the maximum Following this procedure.

Other methods like beag, electrocytes assays QPCR, knockout and or expression strength can be used in order to answer the forthcoming questions After its development. This technique gave the VFR researchers in the field of molecular and cell biology to explore the regulatory networks and mechanisms in RAM positive.