Qiashredders and rneasy mini kit

Total mRNA was obtained from cell pellets using the QIAshredders and RNeasy mini kit (Qiagen). Final mRNA concentration (ng/μl) and quality (260/280 and 260/230 ratio) of the resulting flow though was measured using a Nanodrop 2000.
cDNA was synthesised via reverse transcription, using a Taqman reverse transcription (RT) kit (Life technologies). qRT-PCR was performed on a Biorad i-cycler with a MyIQ detection system, using IQ SYBR Green supermix, 1 μl of template cDNA, and 500nM of forward and reverse primers with the following program:

95 °C – 5 min

40 cycles of:

95 °C – 15 s

60 °C – 30 s

72 °C – 30 s

Florescence was detected as the end of each amplification cycle (step 2c).
Melt curve analysis was performed on all reactions at 0.5 °C increments between 55 and 95 °C to detect any genomic DNA contamination, primer dimers, and/or non-specific amplification. Data were analysed using BioRad IQ software, and the transcript copy number estimated by normalising results to the housekeeping gene (HKG) GAPDH, using the following equation where Ct = cycle threshold.
Copy number of target = 2500*1.93^(HKi – target Ct)
All primers were designed using NCBI Primer-BLAST software, with melting temperatures of 60 °C and primer lengths of ~20. All reactions were performed in triplicate. See Additional file 3 (Table S3) for primer sequences.

Total mRNA was obtained from PICs pellets using the QIAshredders and RNeasy mini kit (Qiagen, Hilden, Germany). Final mRNA concentration (ng/μL) and quality (260/280 and 260/230 ratio) of the resulting flow though was measured using a Nanodrop 2000. cDNA was synthesised via reverse transcription, using a Taqman reverse transcription kit (Life Technologies, Carlsbad, CA, USA). qRT-PCR was performed on a Biorad CFX-connect Real-Time PCR System with a MyIQ detection system, using IQ SYBR Green supermix, 1 μL of template cDNA, and 500 nM of forward and reverse primers with the following program:

95 °C—5 min

40 cycles of:
a.

95 °C—15 s

b.

60 °C—30 s

c.

72 °C—30 s

Primers sequences are listed in Supplementary Table S3. Florescence was detected as the end of each amplification cycle (step 2c). Melt curve analysis was performed on all reactions at 0.5 °C increments between 55 °C and 95 °C to detect any genomic DNA contamination, primer dimers, and/or non-specific amplification. Data were analysed using BioRad IQ software, and the transcript copy number estimated by normalising results to the housekeeping genes (HKG) GAPDH, ß-actin, and B2M. All reactions were run in triplicate.