Total RNA was extracted from cells using Trizol reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s protocol. Total RNA was reverse transcribed with a PrimerScript RT-PCR kit (Takara Biotechnology, Dalian, China). Real time qPCR was conducted using a standard SYBR Green PCR kit (Roche, Upper Bavaria, Germany) protocol with a CFX real-time instrument (Bio-rad, Hercules, CA, USA). The relative expression was calculated using the 2-∆∆Ct method. The transcription level of GAPDH was used as an internal control. All shRNA, siRNA and specific primers are listed in Additional file 1 : Table S1 and Additional file 2 : Table S2.
Primerscript rt pcr kit
Manufactured by Takara Bio
Sourced in China, Japan
The PrimerScript RT-PCR kit is a reagent kit designed for reverse transcription and polymerase chain reaction (RT-PCR) analysis. The kit provides the essential components for performing sensitive and accurate RNA detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Trizol reagent, by Takara Bio (7 mentions)
Sybr green pcr kit, by Roche (6 mentions)
Cfx real time instrument, by Bio-Rad (3 mentions)
7300 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Takara Bio (2 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr machine, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Specific primers, by GenePharma (1 mentions)
Hairpin it mirnas qpcr quantitation kit, by GenePharma (1 mentions)
Abi prism 7700 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Merck Group (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Mirna qpcr kit, by GeneCopoeia (1 mentions)
27 protocols using primerscript rt pcr kit
1
RNA Extraction, Reverse Transcription, and qPCR Analysis
2
Quantification of miR-486 and Its Target Genes
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After the transfection of the cells with the miR-486 mimics/inhibitors, total RNA was purified using Trizol reagent (Invitrogen) following the manufacturer’s instructions. cDNA (complementary DNA) was synthesized using the PrimerScript RT-PCR kit (TaKaRa) according to the manufacturer’s instructions.
The expression of miR-486 and the 5S internal control gene were quantified using real-time PCR quantification and the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer’s protocol. Specific primers for miR-486 and 5S were designed by GenePharma. The expression of these genes was analyzed using a 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
The expression of PTEN, AKT, mTOR, and β-actin was quantified using Real-Time-PCR quantification and SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer’s instructions. β-actin was used as a loading control. The primers that were used for qRT-PCR are listed inTable 1 . All qPCR reactions were performed in triplicate.
The expression of miR-486 and the 5S internal control gene were quantified using real-time PCR quantification and the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer’s protocol. Specific primers for miR-486 and 5S were designed by GenePharma. The expression of these genes was analyzed using a 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
The expression of PTEN, AKT, mTOR, and β-actin was quantified using Real-Time-PCR quantification and SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer’s instructions. β-actin was used as a loading control. The primers that were used for qRT-PCR are listed in
3
Quantitative Analysis of TopBP1 Expression
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Trizol reagent (TaKaRa Biotechnology, Dalian, China) was used for extracting total RNA from cells and total RNA was reverse transcribed with a PrimerScript RT-PCR kit (Takara Biotechnology, Dalian, China) [31 (link)]. The Taqman TM RT-PCR system (Applied Biosystems, Carlsbad, USA) was used to conduct quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) according to the manufacturer’s protocol. The primers used are follows: TopBP1 forward: 5’- TTCAGCAACTCACAGTTAAGCA-3’ and reverse: 5’ GGCACACTCATACTTCTGACC-3’. PCR was performed in the presence of SYBR green (Sigma-Aldrich, St. Louis, USA) using 15 ng of total RNA with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
4
Quantifying miRNA and mRNA Levels
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Total RNA was extracted from cells or human OSCC tumor tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was used for reverse transcription using the PrimerScript RT-PCR kit (TaKaRa Biotechnology, Dalian, China). miRNAs were reverse transcribed using sequence-specific stem-loop primers (Invitrogen). Quantitative RT-PCR was conducted according to the previous description [15 (link)]. Briefly, qRT-PCR was conducted using a standard SYBR Green PCR kit (Roche) protocol with a CFX96 Touch™ Real-Time PCR Detection System (Biorad). The relative expression was calculated using the 2−ΔΔCt method. The transcription levels of GAPDH or U6 were used as an internal control. The specific Primers are listed as follows: GAPDH: forward: 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse: 5′-TGGTGAAGACGCCAGTGGA-3′; HK2: forward: 5′-CAAAGTGACAGTGGGTGTGG-3′; reverse: 5′-GCCAGGTCCTTCACTGTCTC-3′.
5
miRNA Expression Analysis via qPCR
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Total RNA was isolated from samples with TRIzol reagent (Invitrogen, USA) as per the protocol provided. Complementary DNA was synthesized using a Primer-Script RT-PCR kit (TaKaRa, Japan). Amplification of complementary DNA templates was conducted utilizing a miRNA qPCR kit (GeneCopoeia, China). The relative expressions of miR-9 and LINC01116 were normalized, respectively, against U6 and GAPDH. Normalization of relative expressions was performed according to the control level utilizing the comparative 2−ΔΔCt method. Primers used in analyses were all procured from GeneCopoeia.
6
RNA Isolation and RT-qPCR Analysis
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Liver samples were dissected, snap frozen, and stored at −80°C until use. RNA was isolated from using TRIzol reagent (Invitrogen Life Technologies). RNA was reverse-transcribed with the PrimerScript™ RT-PCR kit (Takara) according to the manufacturer's protocol. The relative mRNA levels for specific genes were determined by real-time PCR using SYBR Premix Ex TaqTM (Applied Biosystems). PCR products were quantified using the ABI 7900HT real-time PCR system (Applied Biosystems). Gene expression levels were normalized to the housekeeping gene Gapdh. Real-time PCR was performed as follows: 95°C for 10 min, then 40 cycles of 95°C for 5 s and 60°C for 20 s.
7
Gene Expression Analysis by qPCR and Western Blot
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RNA isolation, quantitative real‐time polymerase chain reaction (qPCR) and Western blotting were performed as previously described [17 (link)]. Briefly, total RNA was extracted using TRIzol Reagent (Takara, Kusatsu, Shiga, Japan). A total of 1 μg RNA was transcribed to complementary DNA (cDNA) using PrimerScript RT‐PCR kit (Takara). The qPCR was conducted using an SYBR Green reaction mix (Vazyme, Nanjing, Jiangsu, China) with a LightCycler 96 System (Roche, Basel, Switzerland). Relative expression was calculated using the 2−ΔΔCt method (Ct, cycle threshold). All specific primers are listed in Supplementary Table S3 .
As for Western blotting, the cells were lysed in RIPA lysis buffer (CWBIO, Beijing, China) with protease and phosphatase inhibitors (CWBIO). After identifying quantities of proteins using Pierce BCA Protein Assay Kit (Invitrogen), the proteins were electrophoresed using SDS‐PAGE and transferred to PVDF membranes (Merck, Burlington, Massachusetts, USA), blocked and incubated with primary antibodies at 4°C overnight. Primary antibodies specific to HSF1, LEF1, MMP9, E2F2, E‐cadherin, N‐cadherin, vimentin, PRMT5, FLAG, GAPDH are listed in Supplementary TableS1 . After incubation, the membranes were incubated with HRP‐conjugated secondary antibodies at 25°C for 1 hour. Protein bands were visualized using enhanced chemiluminescence.
As for Western blotting, the cells were lysed in RIPA lysis buffer (CWBIO, Beijing, China) with protease and phosphatase inhibitors (CWBIO). After identifying quantities of proteins using Pierce BCA Protein Assay Kit (Invitrogen), the proteins were electrophoresed using SDS‐PAGE and transferred to PVDF membranes (Merck, Burlington, Massachusetts, USA), blocked and incubated with primary antibodies at 4°C overnight. Primary antibodies specific to HSF1, LEF1, MMP9, E2F2, E‐cadherin, N‐cadherin, vimentin, PRMT5, FLAG, GAPDH are listed in Supplementary Table
8
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was used for reverse transcription with the PrimerScript RT-PCR kit (TaKaRa Biotechnology, Dalian, China). Quantitative RT-PCR was conducted using a standard SYBR Green PCR kit (Roche) protocol with a LightCycler 480 real-time instrument (Roche). The relative expression was calculated using the 2−ΔΔCt method. The transcription level of GAPDH was used as an internal control. All specific primers are listed in Supplementary Table 3 .
9
Intestinal Total RNA Extraction and qRT-PCR
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Total RNA extraction was performed on 100–150 mg of the distal intestine using 1 ml of Trizol (TRI Reagent solution, Invitrogen, Carlsbad, CA, USA) according to the instructions previously described by Orriss et al. (15 (link)). The RNA quality and quantity were assessed by electrophoresis on 1% agarose gels and spectrophotometric analysis with NanoDrop 2000 (A260:280 nm ratio), respectively. The PrimerScript™ RT-PCR Kit (TaKaRa, Kusatsu, Japan) was used to reverse-transcribe RNA into cDNA according to the manufacturer’s instructions. Specific primers (Table S3
10
Quantitative Assessment of RNA Levels
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Shanghai, China) and was reverse transcribed using the PrimerScript RT-PCR kit (Takara, Dalian, China) according to the manufacturer's instructions. Then, RNA levels were assessed by qRT-PCR using the TaqMan MiRNA Assay Kit (Applied Biosystems). The relative expression of targets was determined in triplicate on an ABI 7500 RT-PCR system (Applied Biosystems). β-Actin or U6 small nuclear RNA (snRNA) was used as a reference gene for normalization of miRNA or mRNA expression. The delta Ct method was used to calculate the relative expression. The primers used in this study are shown in Supplementary Table 1 .
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