RNA Extraction and cDNA Synthesis Protocol

RNA was extracted with Direct-zol™ RNA MiniPrep Kit (Zymo Research, California). cDNA was synthesised from 1 µg RNA with Moloney Murine Leukaemia Virus Reverse Transcriptase (USB) in a 20 μl reaction and 0.1 μl cDNA amplified with Red Hot DNA polymerase (Thermo-scientific) in 10 μl [26 (link), 27 (link)]. Primers designed with Primer-BLAST were synthesised by SIGMA-Aldrich. Amplicons were analysed on 3% agarose gels, stained with GelRed (Cambridge Bioscience, Cambridge, UK).

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Saisana M., Griffin S.M, & May F.E. (2021). Insulin and the insulin receptor collaborate to promote human gastric cancer. Gastric Cancer, 25(1), 107-123.

Publication 2021

Direct-zol™ RNA MiniPrep Kit Red Hot DNA polymerase GelRed

Agarose Cdna Dna polymerase Gels Moloney murine leukaemia virus Primer Reverse transcriptase

Corresponding Organization :

Other organizations : Newcastle University

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Variable analysis

independent variables

RNA extraction method: Direct-zol™ RNA MiniPrep Kit (Zymo Research, California)

dependent variables

CDNA synthesis from 1 µg RNA with Moloney Murine Leukaemia Virus Reverse Transcriptase (USB) in a 20 μl reaction
CDNA amplification with Red Hot DNA polymerase (Thermo-scientific) in 10 μl
Amplicon analysis on 3% agarose gels, stained with GelRed (Cambridge Bioscience, Cambridge, UK)

control variables

CDNA synthesis and amplification reaction volumes
Agarose gel concentration (3%)

positive controls

None specified

negative controls

None specified

Annotations

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