Our protocol improved both throughput and time efficiency for obtaining RT-qPCR data directly from single one or small bulk samples without the need for RNA isolation. Using this protocol, over 9, 000 RT-qPCR results can be obtained in just two days of bench work, a process that would typically take about five weeks using standard 96-well qPCR. This protocol provides a faster, robust and highly sensitive RT-qPCR assay in C.elegans, which is ideal for monitoring the interindividual variability in the gene expression between isogenic worms.
Begin by picking the worms from their bacteria lawn onto a fresh unseeded nematode growth medium plate and allowing the worms to move around the plate for five minutes. While the worms are acclimating, in an RNase-free hood, mix 10 microliters of lysis buffer with one to 100 Dnase I per sample and place the lid of a PCR strip upside down on the platform of a dissecting scope. Next add 10 microliters of the lysis mix to domed PCR tube caps, and scoop the worms from the plate to avoid transferring bacteria into each slot of the lid.
When all of the worms have been transferred, close the caps and spin the tubes for five seconds before placing the tubes in a Dewar flask of liquid nitrogen. After the last five seconds, thaw the tubes in a 40 degree Celsius water bath before repeating the freeze/thaw procedure nine more times. After the last freeze/thaw cycle, mix the lysed samples on a thermal mixer for 20 to 30 minutes at four degrees Celsius and approximately 1800 revolutions per minute.
At the end of the mixing incubation, spin down the samples as demonstrated and add one microliter of freshly thawed stop solution to each tube. For the reverse transcription of bulk warm samples, in an RNase-free hood, prepare an enzyme buffer master mix and add 14 microliters of master mix to 11 microliters of each sample. Run the samples through a thermocycler using the indicated reverse transcription program and dilute the resulting cDNA product at a one to four ratio in nuclease-free water.
For target specific preamplification, add 3.75 microliters of preamplification master mix into one well per sample in a 96-well plate and add 1.25 microliters of each cDNA solution to each well of master mix. When all of the samples have been loaded, cover the plate with sealing tape and briefly vortex the samples before spinning them down by centrifugation, then load the plate onto a thermocycler and run the program as indicated. To remove unincorporated primers from the preamplification, at the end of the thermocycle centrifuge the sample plate and carefully remove the seal.
Add two microliters of exonuclease I master mix to each preamplification reaction and reseal, centrifuge, and load the plate back into the thermocycler. At the end of the cycle, dilute the samples with 18 microliters of Tris-EDTA buffer. To set up a multi-array chip, add 3.6 microliters of assay loading master mix and 0.4 microliters of 50 micromolar forward reverse primer into one well per sample in a 384-well plate.
Then add 2.2 microliters of sample master mix and 1.8 microliters of each preamplified exonuclease I treated sample to the appropriate wells of the plate. To set up a single-array chip, add 5.4 microliters of the assay loading master mix and 0.6 microliters of the forward reverse primer to one well per sample of a second 384-well plate, then add 3.3 microliters of sample master mix and 2.7 microliters of each preamplified exonuclease I treated sample to the appropriate wells of the prepared single-array plate. To prime the nanofluidics chip for the first run, holding the chip at a 45 degree angle slowly and carefully inject 150 microliters of control line fluid into the accumulators of the chip.
When all of the fluid has been loaded, remove the blue protective film from the bottom of the chip and place the chip into the nanofluidics PCR priming machine with the barcode facing outwards. Then run the Prime(153x)script. After priming, place the chip onto a dark surface and as each assay or a sample mix is loaded, remove the corresponding barrier plugs if necessary and add the appropriate volume of assay or sample mix to the corresponding inlets of the nanofluidic chip according to the experimental plan.
After loading, place the chip into the nanofluidic thermocycler and run the appropriate protocol in the data collection software. If the whole multi-array chip is not used, perform a post-script run to allow downstream use of remaining chip arrays. To test if the worm-to-Ct protocol is a valid cDNA extraction method, the method was compared to standard guanidium thiocyanate-phenol-chloroform extraction methods.
Globally, hsp-70 mRNA expression levels per 100 nanograms of total RNA were comparable using both the phenol-chloroform and worm-to-Ct methods. However, in the cases of highest hsp-70 expression, the expression was higher with the worm-to-Ct method, indicating an improved sensitivity. For guanidium thiocyanate-phenol-chloroform extraction of bulk samples, hsp-70 decreased by 82.7 in hsf-1 mutants compared to controls and by 92.3%for warm-to-Ct, indicating that the decrease was comparable between the two methods.
Using cDNA obtained from either bulk samples of 25 worms or from an average of 36 single worm samples, the methods detected comparable expression levels for all of the chaperones tested, indicating that the parameters obtained from single worms are reliable. Here, the mean expression of multiple hsp transcripts from single worms following a short heat shock are shown. As observed, the variability in the expression of the transcripts differ dramatically across different genes.
For every transcript tested using single worm samples, the technical coefficients of variability were lower than the biological coefficients of variability, indicating that technical triplicates was not required for parameter estimation when qPCR was performed on single worms. When performing this protocol, it is essential to pipet small volumes accurately and not to pipet reverse into the nanofluidic chip. This protocol can be used to obtain large sets of RT-qPCR data, which can then be exported and processed as desired using Excel or R scripts.
