Total cellular RNA was extracted with Trizol (TaKaRa, Cat# 9109). Subsequently, complementary DNAs (cDNAs) were synthesized using First-Strand Synthesis System (TaKaRa, Cat# RR036A) according to the manufacturer’s instructions. cDNA was analyzed by qPCR with SYBR Green (YEASEN, Cat# 11143ES50) and gene-specific primers (mPRELID2, forward 5′-GTCGCTTGCTTCCTC-3′, reverse 5′-GATTTTCTACGCTTTCC-3′; mFUNDC2, forward 5′-GCTAACAGTCAAGGAAA-3′, reverse 5′-TCTGGAATACGAAACC-3′; mMFN1, forward 5′-ATCACTGCAATCTTCGGCCA-3′, reverse 5′-AGCAGTTGGTTGTGTGACCA-3′; mSDHA, forward 5′-GAAGATTTATCAGCGTG-3′, reverse 5′-GTGTAAGAGTGAGTGGC-3′; mKi67, forward 5′-GCTCACCTGGTCACCATCAA-3′, reverse 5′-TGACACTACAGGCAGCTGGA-3′; mAFP, forward 5′-GTTTCCAGAACCTGCCGAGA-3′, reverse 5′-CTGAGCAGCCAAGGACAGAA-3′; hHPRT, forward 5′-AGCCCTGGCGTCGTGATTA-3′, reverse 5′-ACAATGTGATGGCCTCCCA-3′; hFUNDC2, forward 5′-ACTGGCAACGAGTGGAGAAG-3′, reverse 5′-CATGCCAAGCAGAAAGCCTC-3′. Relative expression of mRNA was normalized by Succinate dehydrogenase, subunit A (Sdha) or hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA. The real-time PCR results were analyzed and expressed as relative expression of CT (threshold cycle) using the 2−▵▵Ct method.
First strand synthesis system
Manufactured by Takara Bio
Sourced in China, Japan
The First-Strand Synthesis System is a laboratory equipment designed for the synthesis of complementary DNA (cDNA) from a RNA template. It provides the necessary components and protocols for the reverse transcription process, which is a fundamental step in various molecular biology and gene expression analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Takara Bio (1 mentions)
Sybr green, by Yeasen (1 mentions)
Sybr green qpcr master mix, by Selleck Chemicals (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
7500 real time polymerase chain reaction, by Thermo Fisher Scientific (1 mentions)
Takara ex taq, by Takara Bio (1 mentions)
6 protocols using first strand synthesis system
1
Reverse Transcription and qPCR Analysis
2
Quantitative Real-Time PCR Analysis of PDAC Cells
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Total RNA of PDAC cells were isolated with TRIzol Reagent (Invitrogen) and the First-Strand Synthesis System (Takara) was used for first-strand cDNA synthesis. Each sample was processed in triplicate and each experiment was repeated at least three times independently. β-actin was used as loading control. The cDNA was used as template for q-PCR. 2x SYBR Green qPCR Master Mix (Bimake) and CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) were used in the q-PCR experiment. Primer sequences used in this study were shown in Table S2 .
3
Quantifying Gene Expression in Fibroblasts Exposed to Ozone Oil
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Total RNA was extracted from cultured fibroblasts treated with or without ozone oil by TRIzol (15596018, Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized from total RNA with the First-strand Synthesis System (2680A, Takara) according to the manufacturer’s instructions. Real-time PCR was performed by SYBR Premix Ex Taq II (DRR081A, Takara) with 7500 Real-Time PCR system (Applied Biosystems) and normalized to the expression of GAPDH. Relative quantitation and statistics were estimated as the mean of three replicate assays calculated by the 7500 FAST system sequence detection software Q17 (Applied Biosystems). The primers used are:
Collagen I-F: 5ʹ-GCTCCTCTTAGGGGCCACT-3ʹ
Collagen I-R: 5ʹ-CCACGTCTCACCATTGGGG-3ʹ
α-SMA-F: 5ʹ-AGGGAGTAATGGTTGGA ATGG-3ʹ
α-SMA-R: 5ʹ-GGTGATGATGCCGTGTTCTA-3ʹ
TGF-β1-F: 5ʹ-CTCCCGTGGCTTCTAGTGC-3ʹ
TGF-β1-R: 5ʹ-GCCTTAGTTTGGACAGGATCTG-3ʹ
Gapdh-F: 5ʹ-AGGTCGGTGTGAACGGATTTG-3ʹ
Gapdh-R: 5ʹ-TGTAGACCATGTAGTTGAGGTCA-3ʹ.
4
Expression Analysis of IL-35 Subunits
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TRizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from PBMCs. First-strand cDNA was synthesized using the First-Strand Synthesis System (TaKaRa, Dalian, China). SYBR Green PCR Master mix (TaKaRa, Dalian, China) was used to perform the Real-time PCR.
An Applied Biosystems 7500 Real-time Polymerase Chain Reaction (RT-PCR) system was used to analyze the subunit EBI3 and p35 of IL-35. The following primers were employed in each reaction: EBI3, forward 5′-GGCAAGTAGCAAG GGCTTC-3′ and reverse 5′-AGTCGGTCATCTGAGGTTGC-3′; p35, forward 5′-TCCTCCTTGAAGAACCGGA-3′ and reverse 5′-TGA CAACGGTTTGGAGGGAC-3′. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the control: forward 5′-CAGGAGGCCATTGCTGATGAT-3′ and reverse 5′-GAAGGCTGGGGCTCATTT-3′. The thermal cycling conditions are described as follows: Following an initial denaturation step at 95 °C for 30 s, 40 cycles of profile were carried out: 95 °C, 5 s; 60 °C, 34 s. Relative transcripts were determined by the formular: 2−(CTtarget-CTcontrol).
An Applied Biosystems 7500 Real-time Polymerase Chain Reaction (RT-PCR) system was used to analyze the subunit EBI3 and p35 of IL-35. The following primers were employed in each reaction: EBI3, forward 5′-GGCAAGTAGCAAG GGCTTC-3′ and reverse 5′-AGTCGGTCATCTGAGGTTGC-3′; p35, forward 5′-TCCTCCTTGAAGAACCGGA-3′ and reverse 5′-TGA CAACGGTTTGGAGGGAC-3′. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the control: forward 5′-CAGGAGGCCATTGCTGATGAT-3′ and reverse 5′-GAAGGCTGGGGCTCATTT-3′. The thermal cycling conditions are described as follows: Following an initial denaturation step at 95 °C for 30 s, 40 cycles of profile were carried out: 95 °C, 5 s; 60 °C, 34 s. Relative transcripts were determined by the formular: 2−(CTtarget-CTcontrol).
5
Strand-specific RT-PCR for Rtl1 and Rtl1as
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A strand-specific reverse transcriptase reaction was performed for detecting the paternally expressed Rtl1 transcript and the maternally expressed Rtl1as transcript using a SuperScript III First-Strand Synthesis System using the previously reported RT-primer [35 (link)], Rtl1 primer 5′- GGAGCCACTTCATGCCTAAGACGA-3′ and Rtl1as primer is 5′-GTGGAGAACTTCGCTGTCATCGC-3′. Semi-quantitative PCR for both strand-derived transcripts was conducted using TaKaRa Ex Taq (TaKaRa). The PCR results were quantified with the Image J 1.49v software, and the results were normalized to Rpl26 expression. The primers used are listed in Supplementary Table S1 .
6
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and first-strand cDNA was synthesized using a first-strand synthesis system (Takara, Japan) for reverse transcription (RT). cDNA levels were analyzed by qPCR. Each experiment was independently performed in triplicate. The primers used for qPCR listed in Supplementary Table 1 .
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