This method can help answer key questions in the study of host-pathogen interactions by allowing examination of the coding, non-coding, and viral RNA expression involved in the host amino response as well as how a pathogen can affect the host's biological functions. The main advantage of this technique is it presents a protocol optimized for the processing of mRNA and non-coding RNA from globin-reduced RNAseq libraries from a single whole blood sample. Demonstrating the technique will be Sarah Anderson, a technician from my laboratory.
Start by centrifuging the blood tubes at 50 20 times G for 10 minutes at room temperature. Working in a biological safety cabinet, after removing the supernatant, add eight milliliters of RNase-free water to the pellet. Close the tubes and vortex the pellet until it is visibly dissolved, then centrifuge the tubes at 50 20 times G for 10 minutes at room temperature to recover the pellet.
Working in a biological safety cabinet, discard the supernatant and save the pellet. To isolate total RNA, start by pipetting 300 microliters of lysis binding buffer to the pellet. After vortexing, transfer the mixture from each tube to a new labeled 1.5 milliliter centrifuge tube.
Add 30 microliters of homogenate additive from the miRNA isolation kit. Vortex the tubes and place on the ice for 10 minutes. Working in a fume hood, remove the tubes from ice and add 300 microliters of the acid phenol chloroform reagent from the kit and vortex again to mix.
After centrifuging at 10, 000 times G for five minutes at room temperature, carefully remove aqueous phase to a new tube. Based on the amount of aqueous recovery from the previous step, add 1.25 times the volume of 100%ethanol to the aqueous phage in each tube and pipette to mix. Prepare fresh collection tubes containing a filter cartridge for each sample.
Pipette approximately 675 microliters of the lysate-ethanol mixture onto the filter cartridge. Centrifuge briefly at 10, 000 times G to pass liquid through the filter. Discard the flow-through.
Repeat the lysate-ethanol mixture adding to the filter and centrifugation until it has been completely used. Add 700 microliters of wash solution one from the kit to the filter cartridge. Centrifuge briefly to pull the solution through the filters.
Discard the flow-through and keep the same filter cartridges and collection tubes. Add 500 microliters of wash solution two-three to each filter cartridge. After centrifuging again, discard the flow-through and repeat the wash step.
To remove any residual liquid from the filter cartridges, spin them an additional 60 seconds. Transfer the cartridges to fresh collection tubes. Add 100 microliters of 95 degree Celsius preheated nuclease-free water to the center of each filter cartridge.
Centrifuge the cartridges for approximately 20 to 30 seconds at the tabletop centrifuge at maximum speed. To preform hybridization with globin reduction oligos, first denature the RNA by adding each extracted sample into a 0.2 milliliter thin-walled nuclease-free reaction tube. Place the tubes in a thermal cycler at 70 degrees Celsius for two minutes.
After that, immediately place the tubes on ice to obtain optimal RNA quality. While the tubes are cooling, prepare 400 microliters of 10X globin reduction oligo mix and 10X oligo hybridization buffer in a two milliliter tube. To make the hybridization mix, add six micrograms of RNA sample, two microliters of the 10X globin reduction oligo mix, one microliter of 10X oligo hybridization buffer, and nuclease free water to a final volume of 10 microliters to each 0.2 milliliter thin-walled, nuclease-free reaction tube.
Place the tubes in the thermal cycler at 70 degrees Celsius for two minutes. After that, immediately place the tubes on ice. To perform RNase H digestion, first dilute 10X RNase H to one X RNase H with one X RNase H buffer.
Prepare RNase H reaction mix by combining two microliters of 10X RNase buffer, one microliter of RNase inhibitor, and two microliters of one X RNase H, and five microliters of nuclease-free water to a total volume of 10 microliters. Add 10 microliters of the RNase H reaction mix to the globin reduction hybridization samples and mix thoroughly. Digest this reaction at 37 degrees Celsius for 10 minutes and then cool to four degrees Celsius.
Stop digestion by adding one microliter of 0.5 molar EDTA to the tube, and transfer the entire content of the tube to a fresh 1.5 milliliter tube. Immediately after that, add 80 microliters of RNase-free water, 350 microliters of lysis buffer, then 250 microliters of 100%ethanol to each tube and mix well by pipetting. Transfer each 700 microliter sample to a separate elution filter cartridge placed into two milliliter collection tube to collect the flow-through.
Centrifuge for 15 seconds at 8, 000 times G or greater and then discard the flow-through. Place the same elution filter cartridges into new two milliliter collection tubes. To wash the filter cartridge membranes, add 500 microliters of the mild washing buffer to the filter cartridges and centrifuge for 15 seconds at 8, 000 times G or greater.
Discard the flow-through. Then, using the same collection tubes, add 500 microliters of 80%ethanol to the filter cartridges. Centrifuge for two minutes at 8, 000 times G and greater.
Discard both the flow-through and collection tubes, and save the elution spin columns. Place each elution spin column into a new two milliliter collection tube. Centrifuge at full speed for five minutes with the open lid on filter cartridges to dry the spin column membranes and prevent ethanol carryover.
After discarding the collection tubes, place each dried filter cartridge into a fresh 1.5 milliliter collection tube. Add 14 microliters of RNase-free water directly to the center of the filter cartridge membrane. To elute the RNA, centrifuge the tubes for 60 seconds at full speed and then continue with further RNA assessment and preparation.
The globin-depleted sample can now be split and used to prepare the small, non-coding RNA libraries as well as the ribo-depleted mRNA and long, non-coding RNA libraries. After preparing expression libraries of the globin-depleted and ribo-depleted whole blood samples using this protocol, electrophoresis file run summary showed a globin-depleted library sample with RNA integrity numbers that ranged from 6.3 to 9.2. This proved to be an improvement compared to other studies that used globin depletion methods and were only able to achieve RIN numbers at or near six.
Pre-globin-reduction and post-globin-reduction of a single porcine whole blood sample showed that 260 to 280 nanometer concentration ratios are at or above two. Using this protocol, chip-based electropherograms of globin-and ribo-depleted whole blood mRNA library samples prior to pooling and sequencing were obtained. For the mRNA libraries, the representative electropherogram has a peak at approximately 280 base pairs.
For the small ncRNAs representative chip-based electropherograms contain a range of peaks from approximately 100 to 400 base pairs. Peaks at approximately 143 base pairs correspond to miRNAs, and peaks at approximately 153 base pairs correspond to piRNAs. While attempting this procedure, it's important to remember to ice tubes immediately where noted.
Following this procedure, other methods such as next generation sequencing should be performed in order to answer additional questions such as differential expression, epigenetic changes, and their effect on biological pathways and processes. Don't forget that working with phenol-chloroform reagent is extremely hazardous and precautions such as working in a fume hood should be taken. Also, when handling whole blood or infectious organisms, work should be performed in a biological safety cabinet prior to an activation of the infectious organism.
