High capacity cdna reverse transcription kit for mrnas

Manufactured by Thermo Fisher Scientific

The High Capacity cDNA Reverse Transcription kit for mRNAs is a laboratory product designed to convert messenger RNA (mRNA) into complementary DNA (cDNA). This kit provides the necessary reagents and protocols to perform this reverse transcription process efficiently and reliably.

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Lab products found in correlation

Mirvana mirna extraction kit, by Thermo Fisher Scientific (2 mentions) Lightcycler 480 real time pcr system, by Roche (2 mentions)

2 protocols using high capacity cdna reverse transcription kit for mrnas

Quantitative Analysis of miRNA Expression in TNBC

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Total RNA was isolated using the mirVana miRNA extraction kit (Ambion, Thermo Fisher Scientific). 500 ng of total RNA were reverse transcribed using the High Capacity cDNA Reverse Transcription kit for mRNAs (Applied Biosystems, Waltham, MA). Quantitative real-time PCR was performed using a Roche LightCycler 480 Real-Time PCR System (Indianapolis, IN). Each sample was assessed in duplicate and GAPDH or 5S small RNA were used as reference genes for mRNA or miRNA, respectively. The relative quantification of gene expression was calculated using the comparative Ct (2^−ΔΔCt) method, as described in our previous publications (17–19). Note that we could not use this method to determine the relative expression of miR-3189-3p in miRNA-transfected TNBC cells compared to control-transfected cells, since the cycle threshold values (Cts) before transfection were > 35 (Fig. 1).Fig. 1

Relative expression of miR-3189-3p in TNBC after transfection with the miRNA mimic. Bar graphs showing cycle threshold values of mature miR-3189-3p expression in MBA-MD-231 (A), MDA-MB-468 (B), and HME1 (C) cells at 24 and 72 h post-transfection. For reference, expression of the small RNA 5S is also indicated

Vittori C., Jeansonne D., Yousefi H., Faia C., Lin Z., Reiss K, & Peruzzi F. (2022). Mechanisms of miR-3189-3p-mediated inhibition of c-MYC translation in triple negative breast cancer. Cancer Cell International, 22, 204.

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Quantitative RT-PCR for Gene Expression

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Example 2

Quantitative RT-PCR: Total RNA was isolated using the miRVana miRNA extraction kit (Ambion, Austin, Tex., USA). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit for mRNAs or TaqMan assays for microRNAs (Applied Biosystems, Carlsbad, Calif.). Quantitative real-time PCR was performed in duplicate using a Roche LightCycler 480 Real-Time PCR System (Indianapolis, Ind., USA). Each sample was normalized using GAPDH or RNU6B control (ΔCt) and relative quantification of gene expression was calculated using the comparative Ct (2^−ΔΔCt) method, as described in (Eletto et al., (2008) J. Cell. Physiol. 216: 764-770; Pacifici et al., (2013) J. Cell. Physiol. 228: 1070-1075; Rom et al., (2010) FASEB J. 24: 2292-2300 incorporated herein by reference in their entireties). For the clinical samples relative quantification was represented as 1/ΔCt to maintain real differences in Ct values between samples.

US10308939B2. Use of an miRNA to reduce proliferation of a cancer cell (2019-06-04). Board of Supervisors of Louisiana State University and Agricultural and Mechanical College [US]. Inventors: Francesca Peruzzi [US], Duane Jeansonne [US].

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