Hello Everyone. This is Fana Kla from the laboratory of Dr.Xenon Zu, department of Biology at New Mexico State University. We are working on the metagenomics of mosquito gut ecosystem.
Today we are going to demonstrate your A process to deplete ribosomal RNA from the total RNA samples to enrich messenger, RNA for RNA-Seq. RNA-Seq is a widely used technique to study the functions of microbial community. Firstly, I'm going to demonstrate to you how to isolate total RNA and metagenomic, DNA from muscular gut.
Analys Gambia mosquitoes are maintained at an insectory. Mosquitoes are first fed on sugar meal before, and then afterward female mosquitoes are fed on mice for ACHE production. These mosquitoes are fed on sugar and these are fed with mouse blood.
Prior to the procedure, all dissection tools and slides were sterilized. The mosquitoes are surface cleaned with 70%ethanol three times in a Petri dish, and placed back on a glass slide to dry out the ethanol, place the mosquitoes on a different glass slide and put a drop of sterile one XPBS solution. Then gently pull out the gut.
The mosquito guts will be used to get metagenomic DNA and total RNA respectively. Before we begin the RNA work, it is important to take standard precautions to avoid RNA degradation by maintaining a ribonuclease free environment while wearing gloves, clean the work area pipettes, and all required apparatuses with r nay sap. Prior to the procedure, collect 50 guts and place them in a 1.5 milliliter einor tube containing pu reagents.
For total RNA isolation genomic, DNA is isolated from another batch of 50 guts using a metagenomic isolation kit. The concentration of RNA or DNA is measured using NanoDrop. We used our RNA subtraction to remove ribosomal RNA from the total RNA sample.
First, the sample specific RRNA fragments are PCR amplified from the genomic DNA, which we took from the gut samples. The PCR products are purified using a Qiagen PCR purification kit. In this step, the T seven RNA polymerase is used to synthesize antisense RRNA probes.
Set up a reaction using the PCR product from step one and Biotinylated CTP and UTP. Then incubate the reaction overnight at 37 degrees Celsius. Now the antisense RRNA probes are ready to use for RRNA depletion.
This step employs BIOTINYLATED RRNA probes to catch target RRNA in the total RNA sample, the target RRNA and the RRNA probe. Hybrids are then captured by strep dividing coated magnetic beads. Set up the hybridization reaction in a PCR tube and incubate it in a thermal cycler for five minutes at 70 degrees Celsius.
Then ramp down the temperature to 25 degrees Celsius. To allow the RRNA hybridization while the reaction is running, prepare the beads. First, make three aliquots.
Using 100 microliter beads per tube. Wash the beads with one XSSC solution three times. Then leave the tubes on ice.
Once the reaction is ready, diluted by adding 100 microliter one XSSC with 20%form amide. Transfer the reaction solution to the tube with the beads. Mix well and then incubate at room temperature for 10 minutes.
Flicking the tube occasionally, spin the tube briefly and place it on the magnetic rack. Transfer the super natin to a collection tube. Wash the beads two more times each with 100 microliter.
One XSSC buffer solution added. Mix well and then recapture the beads on the magnetic rack. Transfer the supernatants to the collection tube.
Now we have an RRNA depleted RNA sample in the tube. Perform purification using the RN easy minikit from Qiagen. Following the instructions, now we are checking the effectiveness of the depletion.
The RNA samples are loaded onto an Agilent RNA 6, 000 PICO chip and run through the 2100 bioanalyzer. Before subtraction. There were two ribosomal RNA peaks 16 s and 23 s respectively.
After subtraction, the RRNA peaks were effectively removed. The remaining RNA is in the size range from 200 base pair to four kilobase. Now the RNA sample is ready to make a CD NA library for transcriptome sequencing.
We just walk you through the procedure to enrich the messenger, RNA for RNA.Seek. Thank you for watching us.
