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Reverse transcript kit

Manufactured by Takara Bio
Sourced in Japan, China

The Reverse Transcript Kit is a laboratory tool designed to convert RNA molecules into complementary DNA (cDNA) molecules. This process, known as reverse transcription, is a fundamental step in various molecular biology applications, such as gene expression analysis, cDNA library construction, and viral RNA detection.

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16 protocols using reverse transcript kit

1

Analyzing gene expression in spinal cord injury

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Total RNA was extracted from 1.0 cm length of injured cord segments or cultured cell via TRizol (Takara). Then extracted RNA received reverse transcription utilizing a reverse transcript kit (Takara). We performed real‐time PCR for TNFα, iNOS, CD86, IL‐12, IL‐18, CD206, Arginase1, IL‐4, IL‐10, and IRF‐3 with SYBR green (Takara) on Bio‐Rad PCR system (Bio‐Rad). The primers of these genes are listed in Table S1. Gene expression levels were determined by 2−ΔΔCT method and the data were presented normalized by GAPDH.
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2

Real-Time PCR Analysis of Inflammatory Markers

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Total RNAs were isolated from serum samples and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Shanghai, China) as per the instructions. Then, cDNA was reverse synthesized from RNAs using a reverse transcript kit (Takara, Japan). RT‐q PCR was then conducted via SYBR Green PCR Master Mix (Takara, Japan) on a 7500 ABI real‐time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc., Shanghai, China). The expressions of lncRNA MALAT1 and miR‐240 were normalized to that of U6, calculated with the 2−ΔΔCT method. The primers sequences were as follows: IL‐1β (forward), 5′‐ATGATGGCTTATTACAGTGGCAA‐3′, and (reverse) 5′‐GTCGGAGATTCGTAGCTGGA‐3′; IL‐6 (forward), 5′‐ACTCACCTCTTCAGAACGAATTG‐3′, and (reverse) 5′‐CCATCTTTGGAAGGTTCAGGTTG‐3′; TNF‐α (forward), 5′‐CTCTTCTGC CTGCTGCACTTTG‐3′, and (reverse) 5′‐ATGGGCTACAGGCTTGTCACTC‐3′; lncRNA MALAT1 (forward), 5′‐TGTGACGCGACTGGAGTATG‐3′, and (reverse) 5′‐CAAAGGGACTCGGCTCCAAT‐3′; miR‐204 (forward), 5′‐TGGCTACAGTCTTTCTTCA‐3′, and (reverse) 5′‐CTCATGGGACAGTTATGG‐3′; APOL1 (forward), 5′‐TAAGGTACCGACAGAGGGAGGCAGCC‐3′, and (reverse) 5′‐ACCGTCGACTCAGAAGGGTGCCAGACCC‐3′; U6 (forward), 5′‐GCTTCGGCAGCACATATACTAAAAT‐3′, and (reverse) 5′‐CGCTTCACGAATTTGCGTGTCAT‐3′; GAPHD (forward), 5′‐TGCACCACCAACTGCTTAGC‐3′, and (reverse) 5′‐GGCATGGACTGTGGTCATGAG‐3′.
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3

Quantitative RT-PCR of DPC RNA

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Total RNA from DPCs was extracted with Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared with 1 μg total RNA using a reverse transcript kit (Takara). qRT-PCR was performed using the CFX96 Real-Time System (Bio-Rad Laboratories) with SYBR Premix Ex Taq II (Takara). Relative gene expression was analyzed using a 2-ΔΔCt method and normalized to the expression of the housekeeping gene Gapdh.
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4

Mechanical Force-Induced Gene Expression

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Total RNAs were extracted from DPSCs and from PDLSCs after the application of mechanical force using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol, after which cDNAs were synthesized using a reverse transcript kit (Takara Biotechnology, Japan). Real-time PCR was performed using a SYBR Green PCR kit (Takara Biotechnology, Japan) and the sequences of RNA are as follows: GAPDH (5ʹ-CCAAGGAGTAAGACCCCTGG-3ʹ, 5ʹ -AGGGGAGATTCAGTGTGGTG-3ʹ), RANKL (5ʹ-ATCGTTGGATCACAGCACATCAG-3ʹ, 5ʹ-GGATGTCGGTGGCATTAATAGTGAG-3ʹ) and OPG (5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ, 5ʹ-TGGTGAAGACGCCAGTGGA-3ʹ).
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5

Quantitative Analysis of Gene Expression

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Total RNA was prepared from cells, using RNAiso reagent (Takara). RNA was reverse transcribed into cDNA using a reverse transcript kit (Takara) according to the instructions of the manufacturer. Complementary DNA was amplified by using recombinant Taq DNA polymerase (Takara) and specific oligonucleotide primers of PGC‐1β, tumor necrosis factor receptor–associated factor 6 (TRAF6), and β‐actin (Supplementary Table 2, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40868/abstract). SYBR Green–based qPCR was performed using a Roche LightCycler 480 sequence detector system (25).
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6

Quantifying ETS1 Expression in AS Patients

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Total peripheral leukocyte RNA was extracted from 50 AS patients and 161 healthy controls by the TRIzol reagent method (Invitrogen, Carlsbad, CA, USA), then underwent reverse transcription by use of a reverse transcript kit (Takara, Otsu, Shiga, Japan). Allelic-specific expression of ETS1 was analyzed only in healthy controls. RNA samples were treated with RNase free DNAase to eliminate genomic DNA contamination before being reverse-transcribed into cDNA. Real-time quantitative RT-PCR to amplify cDNA involved the Roche 480 real-time PCR system with SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Relative expression was analyzed by the comparative threshold cycle (Ct) method and normalized to that of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calculated by the 2-△△Ct method and log10 transformed. The primer sequences for ETS1 were sense 5′-TACACAGGCAGTGGACCAATC-3′ and antisense 5′-CCCCGCTGTCTTGTGGATG-3′; and GAPDH, sense 5′-CCAGGTGGTCTCCTCTGACTT-3′ and antisense 5′-GTTGCTGTAGCCAAATTCGTTGT-3′. Melting curve analysis was used to confirm specificity, with four duplicate wells used for each subject [16 (link)].
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7

Gene Expression Analysis in Lymphoid Tissues

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The total RNA was isolated from frozen jejunum, spleen, thymus, and bursa, and then the concentration was determined using Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). Following purification, RNA (approximately 200 ng) was used as template for reverse transcriptase reactions using a reverse transcript kit (Takara, Japan). The resulting cDNA was immediately profiled for mRNA expression using a 7900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were selected as the reference genes, and relative gene expression was calculated as previously described [31 (link)]. Primer sequences for all genes are provided in Table S2.
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8

Quantifying mRNA and miRNA Expressions

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Total RNA was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized with a reverse transcript kit (Takara) according to the manufacturer’s instructions. qPCR was performed in a ViiA 7 real-time PCR system (Applied Biosystems) using the SYBR premix EX TAG (Takara). miRNAs were detected by using Bulge-loop primers designed from RiboBio Biotech (Guangzhou, China). mRNA primers were designed and synthesized from Sangon Biotech. β-actin and small nuclear RNA U6 were used as internal controls for mRNAs and miRNAs.
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9

Quantifying Muscle Protein Expression

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RNA from breast muscle was extracted, and then reverse transcribed into cDNA using a reverse transcript kit (Takara, Japan). Beta ryanodine receptors (RYR2), Ca2+-storage protein calsequestrin (CASQ2), μ-calpains (Capn1), m-calpains (Capn2), calpastatin (Cast), and Caspase 3 mRNA were quantified by RT-PCR using a 7900 Fast Real-Time PCR System (Applied Biosystems, USA). Values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. Primer sequences can be found in Table S3.
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10

Quantitative EV71 RNA Detection

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Total RNA was isolated from the samples using the TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using random hexamers with a reverse transcript kit (TaKaRa, China) according to the user manuals. The cDNA was subjected to the EV71 RNA Detection Kit (Shanghai ZJ Bio-Tech Co., Ltd) specific for the VP1 gene. Positive fragments adjusted to a series of concentrations were used as a standard curve.
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