RNA Extraction and cDNA Synthesis

Total RNA was extracted using proteinase K buffer and TRIzol (Invitrogen) as performed previously (Bukhari et al., 2016 (link)). The cDNA was synthesized from 1 μg of RNA using M-MuLV Reverse Transcriptase (NEB) and random hexamer primer (Promega). qPCRs were run on LightCycler^® 480 Instrument II (Roche) using 2 X SYBR green mix (Bio-rad). All primers used are listed in Table S6.

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Bukhari S.I., Truesdell S.S., Datta C., Choudhury P., Wu K.Q., Shrestha J., Maharjan R., Plotsker E., Elased R., Laisa S., Bhambhani V., Lin Y., Kreuzer J., Morris R., Koh S.B., Ellisen L.W., Haas W., Ly A, & Vasudevan S. (2023). Regulation of RNA methylation by therapy treatment, promotes tumor survival. bioRxiv.

Publication Preprint 2023

TRIzol M-MuLV Reverse Transcriptase random hexamer primer LightCycler® 480 Instrument II 2 X SYBR green mix

Buffer Cdna M mulv Primers Promega Proteinase k Reverse transcriptase Sybr green Trizol

Corresponding Organization : Massachusetts General Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Gene expression measured by qPCR

control variables

Amount of RNA used for cDNA synthesis (1 μg)
Reverse transcriptase used (M-MuLV Reverse Transcriptase)
Primer used for cDNA synthesis (random hexamer)
QPCR instrument used (LightCycler® 480 Instrument II)
QPCR reagent used (2 X SYBR green mix)

controls

Positive control: None mentioned
Negative control: None mentioned

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