This procedure begins with acquisition of marine microbial biomass from water samples by pre-filtration through five and three micrometer filters, followed by collection onto a 0.22 micrometer filter. Total RNA is then extracted from the organisms retained on the 0.22 micrometer filter. Our RNA is removed from the total RNA by selective enzymatic degradation and subtractive hybridization enriched Mr.N is amplified linearly via polyA tailing and in vitro transcription.
The amplified antisense RNA is then converted to double stranded CD NA using random hexamer. The resulting CD NA can be cloned and sequenced or directly pyro sequenced. Hi, I'm Rachel Persky from the laboratory of Dr.Marianne Moran at the University of Georgia Department of Marine Sciences.
Today we're going to show you a procedure for looking at gene expression from environmental samples. Using environmental meta transcriptomics. Scott Gifford will show you how to process environmental samples for RNA collection.
I'll show you how to extract RNA from the samples and Johanna RINs. AON will demonstrate RNA removal. Maria Kosta will then go through the RNA amplification and CD NA synthesis steps.
So let's get started. To begin the environmental RNA collection, we set up the filter line system by placing one end of the tubing into the water at the desired depth for sampling and attach the opposite end to a high volume three micrometer filter placing the pump in between the two ends of tubing. The three micrometer filter is then connected to the 0.22 micrometer filter tower and outflow is directed from the 0.22 micrometer tower into a graduated cargo.
Run several liters of water through the system to rinse. When finished, remove the tubing from the water while the pump is running to purge the remaining water from the line. Next, use a pair of forceps to place a 0.22 micrometer filter on the filter tower and close it.
Place the end of the line back into the water and turn on the pump. Allow the air to be purged from both the filters and monitor the volume of water filtered with the on the carboy. Once the desired volume has been filtered, remove the line from the water and burge the remaining water from the system.
As soon as the filter is dry, quickly remove the top filter plate and fold the filter paper. Place the filter into a whirl pack and snap freeze in liquid nitrogen. Before we begin the experiment, it is important to note that because RNAs are ubiquitous and mRNAs degrade rapidly, standard precautions for working in a ribonuclease free environment must be followed, including work surfaces and pipettes.
With this in mind, clean gloves should be worn when handling any materials used in this procedure. And samples should be processed in as short a time as possible. To begin the total RNA extraction from the 0.22 micrometer filter, we prepare bead beading tubes by adding eight milliliters of RLT buffer with beta meto ethanol added to a 50 milliliter conical tube.
Then one to two milliliters of RNA beads from a mobile power soil extraction kit are added. Quickly remove the frozen filter from the freezer and keeping it in the world pack. Shatter it with a mallet.
Add the shattered filter to the prepared 50 milliliter conical tube and seal the tube lid with parfum or lab tape vortex for 10 minutes. Then centrifuge the 50 milliliter tube at 5, 000 RPM for one minute. At room temperature, remove as much of the lysate as possible and transfer it to a 15 milliliter tube before centrifuging again for five minutes.
At 5, 000 times G, the supernatant is then transferred to a new 50 milliliter tube and one volume of 100%ethanol is added to the lysate. Using a 10 milliliter syringe, draw the lysate up through an 18 to 21 gauge needle and pass it back out about five times. Subsequent extraction steps, use the KaiGen R and easy mini kit following the manufacturer's instructions, applying the lysate to the provided column with either the vacuum manifold or with repeated centrifugation.
Quantify the total RNA spectro photometrically. After determining the total RNA yield, we use the Ambien turbo DNA free kit according to the manufacturer's instructions to remove any DNA contaminations. Now that the RNA extractions complete, I'm gonna hand off the total RNA to Johanna, who's gonna do first and second RRNA removal steps.
Thanks Rachel. To begin the RRNA removal gently mix and briefly centrifuge the mRNA only 10 times reaction buffer prior to use in a sterile RNA free 0.5 milliliter tube, combine the appropriate volumes of 10 times reaction buffer RNA inhibitor, total RNA sample and terminator exonuclease. According to the epicenter protocol, incubate the reaction at 30 degrees Celsius for 60 minutes in a thermocycler with a heated lid or water bath.
Next, terminate the reaction with a phenol ethanol precipitation By first adding RNAs free water to a total volume of 200 microliters and then adding one volume of a one-to-one phenol chloroform solution to the mRNA centrifuge. Collect the aqueous phase and then precipitate with a hundred percent ethanol and assault. This is a potential stopping point.
RNA can be stored at negative 80 degrees Celsius until one is ready to use the sample in downstream applications. Once we have completed the first mRNA enrichment, we will use the Ambien microbe enrich and microbe express kits according to the manufacturer's instructions to remove any residual RRNA from our sample. To facilitate the removal of eukaryotic RRNA, we will use the microbe enrich kit.
All volumes are determined by reading the manufacturer's instructions. To begin pipette, 300 microliters of binding buffer into a 1.5 milliliter tube. Add five to 100 micrograms total RNA in a maximum volume of 30 microliters and vortex.
Gently add two microliters of capture oligo mix for five micrograms RNA in binding buffer and tap the tube gently. Briefly spin down the mixture. Denature the mixture at 70 degrees Celsius for 10 minutes before kneeling the mixture at 37 degrees Celsius for one hour.
During this incubation, prepare the oligo mag beads by washing them once in nuclease free water and once in binding buffer and capturing the beads on a magnetic stand between washes. Store the beads on ice and warm them up to room temperature. Five minutes before use heat the wash solution to 37 degrees Celsius in a heat block.
Add the RNA capture oligo mix to the washed oligo mag beads bury gently mix the tube and spin. Briefly incubate the mix at 37 degrees Celsius for 15 minutes. Place the tube in the magnetic stand and wait for about three minutes before aspirating the mRNA containing supernatant and then transferring it to a collection tube on ice.
Next, add 100 microliters of pre warmed wash solution to the captured beads. And gently vortex capture the beads and recover the supernatant fool. This supernatant with Mr.NA in the collection tube on ice.
Place a collection tube on ice while preparing to remove prokaryotic RRNA using the ambi microbe express kit, which we'll use to remove residual prokaryotic, RRNA. Use bioanalyzer or Experion to check for RRNA contamination and MR.NA quality. This is a potential stopping point.
RNA can be stored at negative 80 degrees Celsius. Once the RRNA has been removed, we will use the Ambien message AMP two bacteria kit according to the manufacturer's instructions to amplify our mRNA. So now that we have a pure sample of mRNA, I will hand over the experiment to Maria, who will take you through mRNA amplification and CDNA synthesis.
Thank you, Hannah. Before we begin the mRNA amplification, calculate the volumes of each component of the Ambien message AMP two bacteria kit master mixes online, we begin polyadenylation of template RNA by placing up to 1000 nanograms of total RNA into a sterile RNA free micro fuge tube. Use 6.5 microliters of sample and no RNA free water in the first buffer to synthesize a RNA through in vitro transcription.
It is recommended to use a hybridization oven or incubator because of their uniform temperature. And because it is extremely important that condensation does not form inside the tubes, we strongly recommend a 14 hour in vitro transcription reaction incubation. To maximize a RNA yield for the highest A RNA yield conduct the IBT in a 40 microliter final reaction volume store A RNA at negative 80 degrees Celsius in quats of approximately 10 microliters.
Once the RNA is amplified, it's time to synthesize the complementary or CDNA. The Promega universal ribot Clone. CDNA synthesis system kit is used to synthesize CD NA from the a NA.The kit provides instructions for converting two micrograms, a NA to CD NA.We recommend input of six to 10 micrograms a NA and scaling the reactions accordingly.
Clean the CD NA using either an ethanol precipitation or the promega wizard DNA cleanup system. According to the manufacturer's instructions, store samples at negative 80 degrees Celsius double stranded CDNA can be directly sequenced at this point from 10 liters of seawater, we extracted approximately 10 micrograms of environmental RNA and amplified at 20 fold through conversion of the amplified MR.NA to double stranded CDNA were generated sufficient quantities of material for sequencing and analysis of environmental transcripts. We've just showed you how to assess gene expression from marine microbial communities using environmental meta transcriptomics.
Always remember that when doing this procedure, the mRNA is degrade rapidly and RNAs is are ubiquitous. So take standard precautions for working in a rib nucleus free environment and process the samples as quickly as possible. So that's it.
Thanks for watching and good luck with your experiments.
