Quantitative reverse transcription-PCR

Reverse transcription and real-time qRT-PCR were performed as described, with poly(A) tailing and reverse transcription (RT) for miRNA qRT-PCRs and only RT for mRNAs44 (link). Real-time qRT-PCR was performed with cDNA diluted 1:10 in SsoFast EvaGreen reagents (Bio-Rad) on a CFX96 instrument (Bio-Rad). Expression data were normalised to selected reference genes (PPIA for mRNA and U6, RN7SL, and let-7a for miRNA). Primer sequences can be found in Supplementary Table 1.

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Newie I., Søkilde R., Persson H., Jacomasso T., Gorbatenko A., Borg Å., de Hoon M., Pedersen S.F, & Rovira C. (2016). HER2-encoded mir-4728 forms a receptor-independent circuit with miR-21-5p through the non-canonical poly(A) polymerase PAPD5. Scientific Reports, 6, 35664.

Publication 2016

SsoFast EvaGreen reagents CFX96 instrument

Cdna Genes Mirna Mrna Pcrs Primer Real time pcr Reverse transcription

Corresponding Organization :

Other organizations : Lund University, University of Copenhagen

Top 5 similar protocols

Variable analysis

independent variables

Reverse transcription and real-time qRT-PCR protocol
Poly(A) tailing and reverse transcription (RT) for miRNA qRT-PCRs
Only RT for mRNAs

dependent variables

Expression data

control variables

CDNA diluted 1:10 in SsoFast EvaGreen reagents (Bio-Rad)
Real-time qRT-PCR performed on a CFX96 instrument (Bio-Rad)
Expression data normalized to selected reference genes (PPIA for mRNA and U6, RN7SL, and let-7a for miRNA)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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