Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the selected results from RNA-sequencing (RNA-seq) were verified by RT-qPCR analysis. cDNA was produced using a Prime Script™ RT-qPCR kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR® Premix Ex Taq™ (Takara Bio, Inc.) on a 7900HT fast RT-qPCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with technical triplicates. The gene-specific primers used for RT-qPCR are listed in Table I . Thermocycling conditions were as follows: Initial denaturation at 95˚C for 5 min, followed with 35 cycles at 95˚C for 30 sec and 60˚C for 30 sec. The relative gene expression levels were determined using the 2-ΔΔCq method (31 (link)).
Primescript rt qpcr kit
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript RT-qPCR kit is a reagent kit used for reverse transcription and real-time quantitative PCR (RT-qPCR) analysis. The kit includes the necessary components for the conversion of RNA to cDNA, as well as the reagents required for the subsequent qPCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq, by Takara Bio (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Lightcycler 480 system, by Roche (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
7900ht fast rt qpcr instrument, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Thermo Fisher Scientific (2 mentions)
Sybr premerasetaq kit, by Takara Bio (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Nucleospin rna plant, by Macherey-Nagel (1 mentions)
Rnaeasy plant mini kit, by Qiagen (1 mentions)
Mx3005p qpcr machine, by Eurogentec (1 mentions)
Qpcr core kit for sybr green 1 no rox, by Eurogentec (1 mentions)
Quick amp labeling kit, by Agilent Technologies (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primescript mirna rt qpcr kit, by Sangon (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Sybr green mixture, by Takara Bio (1 mentions)
Sybr premix ex taqtm 2 kit, by Takara Bio (1 mentions)
Trizol reagent, by Solarbio (1 mentions)
15 protocols using primescript rt qpcr kit
1
Quantitative Real-Time PCR Analysis
2
RNA Isolation and qPCR Analysis
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Total RNAs were isolated from cells using the TRIzol reagent(TAKARA). RNAs were reversely transcribed into cDNA with PrimeScript RT-qPCR kit (TAKARA) in a 20 ml reaction. qPCR assays were performed with SYBR premerase Taq kit (TAKARA) on LightCycler96 (Roche) and the sequences of primer pairs used in the present study are shown in Table S2 .
3
Transcript Abundance Analysis Protocol
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For transcript abundance analysis, RNA was extracted from the indicated tissues using the RNAeasy Plant Mini Kit (Qiagen) or NucleoSpin RNA Plant (Macherey-Nagel) according to the manufacturer’s instructions. Isolated RNA was used for microarray or reverse transcription. cDNA was prepared by using the PrimeScript RT-qPCR Kit (TaKaRa). For microarray analysis, extracted RNA was labeled and hybridized according to the protocol of the Quick Amp labeling kit (Agilent, http://www.agilent.com/home ). Agilent single-color technology arrays (60 k) were used for the hybridizations. Raw intensity data were normalized with the quantile method, and subsequently, the low expression probes were discarded after log2 transformation. Differentially expressed probes were filtered after pairwise comparisons (|log2FC| ≥ 1, FDR ≤ 0.05).
RT-qPCR was performed on a Stratagene Mx3005P qPCR machine using a SYBR Green containing reaction mix (Eurogentec, qPCR Core kit for SYBR Green I No ROX). For all qPCR analysis, N. attenuata IF-5A housekeeping gene was used as internal reference. Primer sequences were listed in theS2 Table .
RT-qPCR was performed on a Stratagene Mx3005P qPCR machine using a SYBR Green containing reaction mix (Eurogentec, qPCR Core kit for SYBR Green I No ROX). For all qPCR analysis, N. attenuata IF-5A housekeeping gene was used as internal reference. Primer sequences were listed in the
4
RNA Extraction and qRT-PCR Analysis
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Trizol kit (Invitrogen) was selected for total RNA extraction. The quality and concentration of RNA was tested by UV-Vis spectrophotometry (ND-1000, NanoDrop).
For measuring messenger RNA (mRNA) expression, reverse transcription was processed using PrimeScript RT-qPCR kit (TaKaRa, Dalian, China). SYBR Premix Ex TaqTM (TaKaRa) was adopted for RT-qPCR on the LightCycler 480 system (Roche Diagnostics, Pleasanton, California, USA). GAPDH was used as a normalizer for mRNA. The primers (online supplemental table S1 ) were designed and provided by Shanghai General Biotechnology. The 2–ΔΔCt method was adopted to quantify relative expression levels of target genes.
For measuring messenger RNA (mRNA) expression, reverse transcription was processed using PrimeScript RT-qPCR kit (TaKaRa, Dalian, China). SYBR Premix Ex TaqTM (TaKaRa) was adopted for RT-qPCR on the LightCycler 480 system (Roche Diagnostics, Pleasanton, California, USA). GAPDH was used as a normalizer for mRNA. The primers (
5
Retinal Total RNA Extraction and RT-qPCR
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Total RNA was extracted from intact retina using a traditional method. Retinas were placed in 200 μl of chloroform and 1 ml RNAiso Plus (Takara, 9108, Japan), shaken vigorously, and centrifuged at 12,000 rpm for 5 min (4 °C). The supernatant was transferred to a sterile EP tube, mixed with 500 μl isopropanol, and stored at -20 °C overnight. The solution was centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatant was discarded. Next, 1 ml of 75% alcohol was added to each tube, samples were centrifuged at 7500 rpm for 5 min at 4 °C, and the supernatant was removed. After another centrifugation step at 12,000 rpm for 15 s, the supernatant was removed. Samples were dried at room temperature until alcohol had fully evaporated. Then 20 μl DEPC water was added to dissolve RNA. The RT-qPCR assays were performed using a Prime Script RT-qPCR Kit (Takara) on a Real-Time instrument (Bio-Rad). The Primers used in this study are listed in Additional file 1 : Table S3.
6
Reverse Transcription and qPCR Analysis
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TRIzol-extracted RNA was reverse transcribed into cDNA using the PrimeScript™ RT-qPCR Kit (Takara, Mountain View, CA). RT-qPCR was conducted using SYBR Premix Ex Taq™ (Takara) on LightCycler 480 system (Roche Diagnostics, Pleasanton, CA). As normalized to Actin, fold changes were evaluated using the 2−ΔΔCt method [22 (link)]. Primers were provided by Shanghai General Biotechnology Co., Ltd. (Shanghai, China), with sequences listed in Supplementary Table 1 .
7
Quantitative Analysis of RNA Levels
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Total RNA was extracted from cells, Exo or tissues using TRIzol® reagents (15,596,018, Invitrogen), while lncRNA and mRNA levels were detected by reverse transcription using the PrimeScript™ RT-qPCR kit (RR047A, Takara, Japan). Reverse transcription was performed to detect miRNA levels using the PrimeScript™ miRNA RT-qPCR kit (B532451, Sangon Biotech, Shanghai, China). The SYBR® Premix Ex Taq™ II kit (RR820A, Takara) was used for sample configuration and samples were subjected to real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference for lncRNA and mRNA while U6 was used as an internal reference for miRNA. The primers used for amplification were provided by General Biotechnology (Shanghai, China). Primer sequences are listed in Supplementary Table 1 . The relative transcript levels of the target genes were calculated using the relative quantification method (2-ΔΔCT method), in which mRNA relative transcript levels of the target genes = 2-ΔΔCt. Three replicate wells were set up for each sample and each set of experiments was repeated 3 times.
8
Quantifying Gene Expression Using qPCR
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Total RNA was extracted from tissues and cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse-transcribed into cDNA using a PrimeScript™ RT-qPCR kit (Takara Bio, Inc.) according to the manufacturer's protocols at 37°C for 75 min. qPCR was subsequently performed using SYBR Green mixture (Takara Bio, Inc.). The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 15 sec and 60°C for 35 sec. Relative expression was calculated using the 2−ΔΔCq method (21 (link)). Relative expression levels were normalized to the internal reference genes U6 or GAPDH. The primer sequences used were as follows: lnc-MBI-52 forward (F), 5′-GTCCAGGGACCTCTGACCTA-3′ and reverse (R), 5′-CTGGAGAATCACCCCGACTG-3′; miR-466g F, 5′-CACTAGTGGTTCCGTTTAGTAG-3′ and R, 5′-TTGTAGTCACTAGGGCACC-3′; α-SMA F, 5′-CACCATCGGGAATGAACGCTTC-3′ and R, 5′-CTGTCAGCAATGCCTGGGTA-3′; Col-1 F, 5′-GGTCATTCTCTTCGCAGACAG-3′ and R, 5′-CCACCGGATACTTGGTCTCCA-3′; SMAD4 F, 5′-CTCATGTGATCTATGCCCGTC-3′ and R, 5′-AGGTGATACAACTCGTTCGTAGT-3′; U6 F, 5′-CTCGCTTCGGCAGCACA-3′ and R, 5′-AACGCTTCACGAATTTGCGT-3; and GAPDH F, 5′-AAGGTGAAGGTCGGAGTCA-3′ and R, 5′-GGAAGATGGTGATGGGATTT-3′.
9
Quantitative Analysis of Sestrin 1 Expression
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Total cellular RNA was extracted using TRIzol reagent (Takara Biotechnology, Dalian, China). cDNA was synthesized with the PrimeScript RT-qPCR Kit (TaKaRa) following manufacturer’s protocols. Real-time qPCR was used to analyze gene expression with SYBR Premix Ex Taq (Takara) on an ABI 7500 Real-time PCR instrument. Actin was used as an internal control. Relative gene expression was quantified using the 2-ΔΔCt method. Primer sequences were as follows: Actin: forward, 5′-GTCCACCGCAAATGCTTCTA-3′; reverse, 5′-TGCTGTCACCTTCACCGTTC-3′; Sestrin 1: forward, 5′-ATACCGAGTCTTCGGATGGG-3′; reverse, 5’-AATCTGCTTGGTCCCTGTCC-3′.
10
RNA Extraction and RT-qPCR Analysis
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Total RNA extraction was conducted in GC cells or EVs by TRIzol® reagent (15596-018, Solarbio, Beijing, China) before cDNA synthesis. To detect lncRNA and mRNA expression, a PrimeScript™ RT-qPCR Kit (RR047A, TaKaRa, Tokyo, Japan) was applied for reverse transcription. For miRNA detection, Poly(A) Tailing Kit (B532451, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China; containing universal PCR primer R) was used for reverse transcription to obtain the cDNA of miRNA with PolyA tail. RT-qPCR was conducted on LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA) using SYBR® Premix Ex TaqTM II Kit (DRR081, Takara). Primers for amplification were provided by Shanghai General Biotechnology Co., Ltd. (Shanghai, China; Supplementary Table 2 ). The 2−△△CT method was employed for calculation of the relative transcription levels of target genes that were normalized to GAPDH (for lncRNA and mRNA) and U6 (for miRNA). All investigations involved at least 3 wells, each repeated in triplicate.
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