Real-time qRT-PCR analysis

1.    Grow flies at 25°C.

2.    Upon hatching, collect 1-2 days old flies and place at 18, 25 or 29°C for three days.

3.    Snap freeze the flies in 15ml tubes, using liquid nitrogen, at the desired timepoints, in our case: ZT3, ZT7, ZT11, ZT15, ZT19, and ZT23.

4.    To separate the heads from the bodies, perform three rounds of 5-10 seconds vortexing and back to liquid nitrogen. Collect 20-30 fly heads by passing the contents of the vortexed 15ml tube through a sieve with a 710 µm and a 425 µm mesh. This will allow to separate the heads cleanly as the bodies will be stuck in the first mesh; the heads, in the second, and pieces of legs and wings will pass to the bottom. CRITICAL: keep the sieves at -80 °C and take out immediately before their use.

5.    Extract total RNA using TRI Reagent (Sigma).

            a.    Homogeneize the fly heads with 50 µl of Trizol using a pestle. Repeat homogeneization after adding 150 µl more of Trizol to the sample (total 200 µl of Trizol).

            b.    Add another 800 µl of Trizol to a total of 1ml and mix.

            c.     Add 200 µl of chloroform and vortex thoroughly.

            d.    Incubated at room temperature for 12 min.

            e.    Spin for 15 min (12000 x g, 4 °C).

            f.      Transfer aqueous (top) phase to a fresh tube.

            g.    Add 1 µl of glycogen and 1:1 ratio for Molecular grade isopropanol.

            h.    Keep at -20 °C overnight.

            i.      Spin for 30 min (21000 x g, 4 °C).

            j.      Pipet off supernatant and wash pellet with 75% Ethanol followed by a 10 min spin at maximum speed.

            k.     Pipet off 75% ethanol completely and let dry.

            l.      Resuspend in 20 µl of nuclease free water.

6.    Treat the RNA with DNase I (NEB) following the manufacturer's protocol.

7.    Synthesize cDNA from 1 µg of RNA using iScript kit and oligodT or random primers.

8.    Dilute cDNA 1:60 in nuclease free water.

9.    Design primer pairs to target your gene of interest. In our case, primer pairs of tim isoforms generated by intron retention were designed with the forward primer in the exon and the reverse in the intron that is retained. Primer pairs to depict tim isoforms produced by splicing have one of the primers in the exon junction and the other in one of the exons. Primer3Plus software was used to pick optimum primers with 100-150 bp and a similar temperature of melting (~55 °C).

10.  Perform the quantitative real-time PCR using SYBR green (Bio-Rad) in a C1000 Thermal Cycler Bio-Rad.

            a.     In each well, mix 4 µl of SYBR, 1 µl of primer pair mix at a 10 μM concentration and 3 µl of the diluted cDNA. Prepare at least one negative control per primer pair, in which you add nuclease free water instead of cDNA.

            b.    The PCR mixture was subjected to 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 55 °C for 10 s and 72 °C for 30 s followed by a melting curve analysis.

            c.     Fluorescence intensities were plotted versus the number of cycles by using an algorithm provided by the manufacturer (CFX Maestro Software, Bio-Rad).

 Primer pairs used for amplifying each isoform and as controls:

* / represents the junction of the two exons involved in the generation of the isoform of interest.

Software used:

·         Primer3Plus: Andreas Untergasser, Harm Nijveen, Xiangyu Rao, Ton Bisseling, René Geurts, and Jack A.M. Leunissen: Primer3Plus, an enhanced web interface to Primer3 Nucleic Acids Research 2007 35: W71-W74; doi:10.1093/nar/gkm306

·         CPX Maestro Software: Biorad. 2020, July. Retrieved from: https://www.bio-rad.com/en-us/category/qpcr-analysis-software?ID=42a6560b-3ad7-43e9-bb8d-6027371de67a&WT.mc_id=171004022632&WT.srch=1&WT.knsh_id=4d17ec40-9a85-4550-bfc1-52f0e89b865c&gclid=EAIaIQobChMIz8ixiMGu6gIVNPC1Ch2mxQtDEAAYASAAEgLMsvD_BwE