Using this protocol to remove the inter-sample variation can offer insights into the differential regulation of macromolecules based in the intra-sample discrepancies between mRNA and protein levels. Using the same sample to isolate DNA, RNA, and protein is an attempt to reduce variability, improve reproducibility, and facilitate interpretation. Benefits include saved time and resources at time of sample collection and cross-sectional analysis of valuable and limited samples.
To begin, seed 1, 000 worm eggs in one 10 centimeter plate with appropriate growth conditions. Incubate at 20 degrees Celsius for 72 hours. Then, wash the plate with 5 milliliters of M9 buffer and transfer 1, 000 adult worms into a tube.
Centrifuge the worms at 1, 000 g for one minute, discard the supernatant, and transfer the pelleted worms, with one milliliter of M9 buffer, to a 1.5 milliliter microcentrifuge tube. Centrifuge again at 845 g for one minute and discard most of the supernatant. Store the pelleted worms at minus 80 degrees Celsius for more than four hours.
Next, remove any supernatant from the thawed pellet. Add one milliliter of cold GTCP reagent, mix well by pipetting up and down and place the sample on ice for 10 minutes. Then, add 200 microliters of cold chloroform.
Shake the tube vigorously for 15 seconds and leave it at room temperature for three minutes. Next, centrifuge the tube at 13, 500 g at four degrees Celsius for 15 minutes. With a micro-pipette, transfer the clear top layer into a new RNase-free 1.5 milliliter micro-centrifuge tube.
Transfer the cloudy white interphase layer to a new tube labeled B2 and transfer the pink layer, from the remaining pellet, into a new tube labeled B and place both on ice. Plainly separating the three layers can be difficult. I recommend transferring the cloudy interphase layer into it's own tube rather than precipitating the DNA from this layer from the organic, or pink layer.
To isolate RNA, first add 500 microliters of 100%isopropanol to tube A to precipitate the RNA. Then, incubate the tube at room temperature for 10 minutes. After incubation, centrifuge the tube at 13, 500 g at four degrees Celsius for 10 minutes.
Next, discard the supernatant and add one milliliter of 75%ethanol to tube A to wash the pellet. Centrifuge the tube at 5, 300 g at four degrees Celsius for five minutes. Discard the supernatant and let the pellet air dry for five to 10 minutes.
Add 50 microliters of RNase-free water to reconstitute the pellet of RNA and incubate the pellet at 55 to 60 degrees Celsius for 10 minutes to dissolve it. Finally, use a spectrophotometer to measure the concentration of the isolated RNA at 260 nanometers and to identify any impurities at 230 and 280 nanometers. To isolate the DNA, first add 300 microliters of 100%ethanol to the organic phase in tube B and to the cloudy white layer in tube B2 to precipitate the DNA, mix by inversion and leave the tubes at room temperature for two to three minutes.
Then, centrifuge tubes B and B2 at 376 g at four degrees Celsius for five minutes to pellet DNA. Combine the supernatant from tubes B and B2 in a new two milliliter tube labeled C and leave it on ice for subsequent protein isolation. Next, wash the DNA pellet in tube B2 with one milliliter of 0.1 molar sodium citrate in 10%ethanol for 30 minutes.
Centrifuge tube B2 at 376 g at four degrees Celsius for five minutes. Repeat the wash step and then re-suspend the pellet in 1.5 milliliters of 75%ethanol and leave it at room temperature for 20 minutes. Next, centrifuge tube B2 at 376 g at four degrees Celsius for five minutes and then discard the supernatant and allow the pellet to dry for five to 10 minutes.
Dissolve the pellet in 150 microliters of eight milliliter sodium hydroxide. Then, centrifuge the sample at 376 g at four degrees Celsius for five minutes. Finally, with a micropipette, transfer the supernatant containing the DNA to a new tube.
Use a spectrophotometer to measure the concentration of the isolated DNA at 260 nanometers and to identify any impurities at 230 and 280 nanometers. To precipitate the protein, first add up to 1.5 milliliters of 100%isopropanol to the pink supernatant in tube C, mix by inverting several times, and incubate at the room temperature for 10 minutes. Then, centrifuge tube C at 13, 500 g at four degrees Celsius for 10 minutes.
Discard the supernatant and wash the pellet with two milliliters of 0.3 molar guanidine hydrochloride in 95%ethanol for 20 minutes at room temperature. Centrifuge the tube again at 5, 300 g at four degrees Celsius for five minutes and repeat the wash step as before. Transfer the protein pellet to a new 1.5 milliliter tube labeled C2 and add up to 1.5 milliliters of 95%ethanol, vortex, and let it sit at room temperature for 20 minutes.
Centrifuge the tube at 5, 300 g at four degrees Celsius for five minutes. Discard the supernatant and let the pellet dry at room temperature for 10 minutes. Then, dissolve the pellet in 300 microliters of 5%SDS at 50 degrees Celsius for 60 minutes.
Finally, centrifuge the tube at 13, 500 g at 17 degrees Celsius for 10 minutes. And transfer the supernatant containing the protein to a new tube. To perform a quantitative reverse transcription PCR.
First, use one nanogram of the isolated RNA to prepare cDNA by reverse transcription. To make a stock plate of cDNA, add 100 microliters of one to 100 dilutions of each cDNA sample to individual wells of a 96 well plate, include appropriate controls, such as water-only wells and serially diluted samples, to establish primer efficiency for each gene. To make a master mix for each set of primers, add up to seven microliters of water, a DNA-intercalating cyanide dye, and five micro-molar each of forward and reverse primers.
Add seven microliters of the master mix to the appropriate wells of an RT-qPCR plate. Then, add three microliters of the cDNA from the stock plate and run the plate using a RT-qPCR protocol suitable for the primers being used. RT-qPCR analysis of isolated mRNAs from four independent samples of three worm strains were done to confirm the targets identified by RNA seq assay.
F07C4.12 gene was upregulated in both assays in eat-2 worms, but its upregulation in rsks-1 worms was not detected by RT-qPCR assay. Also, RNA seq detected expression changes of mrp-1 in eat-2 and rsks-1 worms were confirmed via RT-qPCR. Upregulation or down-regulation of organelle marker genes in mutant worms were also detected by RT-qPCR analysis.
Comparison of GTCp versus RIPA-extracted protein in worms separated by SDS page and stained with coomassie blue showed a similar quality with better resolution of larger proteins for GTCp-extracted proteins. Western blot analysis showed similar protein levels in most cases;however, the proteins larger than 75 kilodalton showed lower levels in the RIPA-extracted protein. The comparison between targets mRNA and protein levels from four individual samples of three worm strains showed a low variability of the mRNA levels with greater variability at the protein level.
In the context of DNA isolation, yield is highly dependent on the proficiency to recover the cloudy interphase layer from the organic, or pink, layer. To improve solubilization of proteins from the pellet increasing the volume of resolubilization buffer or adding other detergents besides SDS may be necessary. Using this method can help correctly identify cases where translation of mRNA to protein is not corelative and can lead to deeper investigation of post-transcriptional and post-translational regulatory mechanisms under various conditions.
In our lab, this protocol has been used to conserve valuable and limited time core samples. Furthermore, I can imagine this would be a useful protocol to adopt in the circadian rhythm field. The GCTP reagent and solvents used in the RNA isolation are hazards and should be used with caution.
