High capacity cdna reverse transcription protocol

Manufactured by Thermo Fisher Scientific

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The High-capacity cDNA reverse transcription protocol is a laboratory procedure designed to facilitate the synthesis of complementary DNA (cDNA) from RNA templates. This protocol enables the efficient conversion of RNA molecules into their corresponding cDNA counterparts, which can be utilized in various downstream applications such as gene expression analysis, qPCR, and library preparation.

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Lab products found in correlation

Sybr green reagent, by Thermo Fisher Scientific (6 mentions) Tri reagent, by Merck Group (2 mentions) Rneasy protocol, by Qiagen (1 mentions)

8 protocols using high capacity cdna reverse transcription protocol

Gene Expression Profiling by qRT-PCR

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One microgram of extracted RNA was used to generate complementary DNA (cDNA) using the high‐capacity cDNA reverse transcription protocol (Applied Biosystems). q‐PCRs were then performed using 1/40 of the total of cDNA, 100 nM primer, and SYBR Green reagent following the manufacturer's protocol (Applied Biosystems). Reactions were normalized to ACTA1 as a loading control.
qRT–PCR primers used:
ACTA1 (F: TGCAGAAAGAGATCACCGC, R: CCGATCCACACCGAGTATTTG)
BAX (F: CTGACGGCAACTTCAACTGG, R: GATCAGTTCCGGCACCTTGG)
CDKN1A (F: TGTCACTGTCTTGTACCCTTG, R: GGCGTTTGGAGTGGTAGAA)
DDB2 (F: TCATTGTTGTGGGCCGATAC, R: TGGCTCCAGATGAGAATGT
KDM4A (F: CGGCCAAGTCTATGGAGCC, R: TCATTGAAGCGCATGTCTGAG)
MDM2 (F: TGCCAAGCTTCTCTGTGAAAG, R: TCCTTTTGATCACTCCCACC)
PPDM1D (F: ATAAGCCAGAACTTCCCAAGG, R: TGGTCAATAACTGTGCTCCTTC).

Jiménez A., Lu D., Kalocsay M., Berberich M.J., Balbi P., Jambhekar A, & Lahav G. (2022). Time‐series transcriptomics and proteomics reveal alternative modes to decode p53 oscillations. Molecular Systems Biology, 18(3), e10588.

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RNA Extraction, cDNA Synthesis, and qPCR

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Total RNA was extracted using the RNeasy protocol (Qiagen). RNA concentration was determined by
measuring absorbance at 260 nm. Equal RNA levels were used to generate complementary DNA
using the high-capacity cDNA reverse transcription protocol (Applied Biosystems). Quantitative PCR
was performed using reaction mixtures of 8.4 ng total RNA, 100 nM primer, and SYBR
Green reagent (Applied Biosystems).

Gaglia G, & Lahav G. (2014). Constant rate of p53 tetramerization in response to DNA damage controls the p53 response. Molecular Systems Biology, 10(10), 1-8.

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Quantitative RT-PCR Protocol for Gene Expression

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2 μg of total RNA was used to generate complementary DNA (cDNA) using the high-capacity cDNA reverse transcription protocol (Applied Biosystems). q-PCRs were then performed using 1/200 of the total of cDNA (per reaction in a 384-well plate), 300 nM primer, and SYBR Green reagent following the manufacturer’s protocol (Applied Biosystems). Reactions were normalized to HPRT as a loading control. qPCR primers used are listed in key resources table.^{45 (link)}

Eghbali B., Kessler J.A, & Spray D.C. (1990). Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.. Proceedings of the National Academy of Sciences of the United States of America, 87(4), 1328-1331.

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Quantitative RT-PCR Analysis of Gene Expression

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Same RNA samples as used RNA sequencing were used to generate complementary DNA using the high-capacity cDNA reverse transcription protocol (Applied Biosystems). Quantitative RT-PCR was then performed using 10ng of cDNA, 100 nM primer, and SYBR Green reagent (Applied Biosystems). Normalization was done to the average of both ACTB and GAPDH genes.
qPCR Primers used:

CDKN1A mRNA

FW: TGTCACTGTCTTGTACCCTTG

REV: GGCGTTTGGAGTGGTAGAA

CDKN1A pre-mRNA

FW: CCCGGCCAGGTAACATAGT

REV: CATGGGTTCTGACGGACATC

DDB2 mRNA

FW: TCGTCAGGACCCTCCAC

REV: CGCCAAGGATGTAGCCC

DDB2 intron

FW: CGCTAGAGTGCAGTGATTCG

REV: GGTGGTAGGTGCATGTGGTT

RPS27L mRNA

FW: CGTCCTTGGAAGAGGAAAAG

REV: ACCGTGGTGATCTTGTAGCA

RPS27L pre-mRNA:

FW: GGGATTGCTAGTGTGGTGTG

REV: TGTCCCTGACATTTCCAATTC

GAPDH:

FW: ACATCGCTCAGACACCATG

REV: TGTAGTTGAGGTCAATGAAGGG

ACTB:

FW: ACCTTCTACAATGAGCTGCG

REV: CCTGGATAGCAACGTACATGG

Hafner A., Stewart-Ornstein J., Purvis J.E., Forrester W.C., Bulyk M.L, & Lahav G. (2017). p53 pulses lead to distinct patterns of gene expression albeit similar DNA binding dynamics. Nature structural & molecular biology, 24(10), 840-847.

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Quantitative RT-PCR Protocol for Gene Expression

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Chen J.Y., Hug C., Reyes J., Tian C., Gerosa L., Fröhlich F., Ponsioen B., Snippert H.J., Spencer S.L., Jambhekar A., Sorger P.K, & Lahav G. (2023). Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction. Cell reports, 42(3), 112252.

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qRT-PCR Analysis of p21 Expression

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RNA was collected using the TRIZOL reagent and purified using the Zymo RNA Clean-up kit. cDNA was generated using the high-capacity cDNA reverse transcription protocol (Applied Biosystems). Quantitative RT-PCR was then performed using 8.4 ng total cDNA, 100 nM primer, and SYBR Green reagent (Applied Biosystems). Normalization was done to the GAPDH gene.
qPCR primers used:
p21 Forward: GACTCTCAGGGTCGAAAACG
p21 wild type Reverse: AAGATGTAGAGCGGGCCTTT
p21 mCherry MS2 Reverse: CATGTTATCCTCCTCGCCCT
GAPDH Forward: ACATCGCTCAGACACCATG
GAPDH Reverse: TGTAGTTGAGGTCAATGAAGGG

Hafner A., Reyes J., Stewart-Ornstein J., Tsabar M., Jambhekar A, & Lahav G. (2020). Quantifying the Central Dogma in the p53 pathway in live single cells. Cell systems, 10(6), 495-505.e4.

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Chlamydomonas RNA Isolation and cDNA Synthesis

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Early log-phase cells of Chlamydomonas reinhardtii cc-125 were measured by taking absorbance at 750 nm and counting (OD₇₅₀ = 0.25 − 0.35; 2 × 10⁶ cells/mL). The cells were used to isolate total RNA by the TRI Reagent (Sigma-Aldrich, Rehovot, Israel) protocol. The cDNA was synthesized using the high capacity cDNA reverse transcription protocol (Applied Biosystems, Foster City, CA, USA) with 1 µg RNA as a template. Bacterial clones for recombinant protein expression were generated as described in the Supporting methods S1, S2 and, S3 section and the primers used are listed in Tables S2 and S3.

Amiya R, & Shapira M. (2021). ZnJ6 Is a Thylakoid Membrane DnaJ-Like Chaperone with Oxidizing Activity in Chlamydomonas reinhardtii. International Journal of Molecular Sciences, 22(3), 1136.

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RNA Isolation and cDNA Synthesis

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Early log-phase cells (OD750 = 0.25-0.35, 2×10 6 cells/ml) were used to isolate total RNA by using TRI Reagent (Sigma) protocol. The cDNA was synthesized using High capacity cDNA reverse transcription protocol (Applied Biosystems) with 1 µg RNA as a template. Bacterial clones for recombinant protein expression were generated as described in the supplemental methods section and the primers used were mentioned in supplementale tables 2 and 3.

Amiya R., & Shapira M. (2020). ZnJ6 is a DnaJ-like Chaperone with Oxidizing Activity in the Thylakoid Membrane inChlamydomonas reinhardtii.

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