We performed qRT-PCR analysis as previously described [48 (link),49 (link),50 (link)]. Briefly, total RNA was extracted by Total RNA Isolation Reagent (BS258A, Biosharp, Shanghai, China) and reverse transcribed into complementary DNA (cDNA) by the First-strand cDNA Synthesis Kit (K1072, Apexbio, Huston, TX, USA). Expression of genes was quantified by the SYBR Green PCR Mix (BL697A, Biosharp, Shanghai, China) on the LightCycler 480 (Roche, Basel, Switzerland) instrument. The primer sequences can be found in Table S4 .
First strand cdna synthesis kit
Manufactured by Apexbio
Sourced in United States
The First-Strand cDNA Synthesis Kit is a laboratory tool used for the reverse transcription of RNA to complementary DNA (cDNA). The kit provides the necessary reagents and protocols to convert RNA into single-stranded cDNA, which can then be used for various downstream applications, such as PCR amplification and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (2 mentions)
Sybr green fast mastermix, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr mix, by Biosharp (1 mentions)
Total rna isolation reagent, by Biosharp (1 mentions)
Total rna extraction kit, by Solarbio (1 mentions)
Trizol rna isolation reagent, by Thermo Fisher Scientific (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Rna clean concentrator kit, by Zymo Research (1 mentions)
Dnase 1, by Zymo Research (1 mentions)
Quick rna fungal bacterial miniprep kit, by Zymo Research (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Total rna extraction kit, by Promega (1 mentions)
Saos 2, by American Type Culture Collection (1 mentions)
9 protocols using first strand cdna synthesis kit
1
Quantitative Real-Time PCR Analysis
2
Quantifying gene and miRNA expression
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Total RNA was extracted from OC cell lines and tissues by total RNA Extraction Kit (Solarbio Science & Technology, Beijing, China), followed by synthesizing to cDNA using First-Strand cDNA Synthesis Kit (APExBIO Technology, Houston, TX, USA). qRT-PCR was then performed with SYBR Green FAST Mastermix (Qiagen). The 2-ΔΔCT method was utilized to calculate the relative expression. GAPDH (for PART1 and FOXK1) or U6 (for miR-503-5p) were used as the internal controls.
3
Quantifying CRC Cell Total RNA
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Total RNA of CRC cells was extracted with TRIzol RNA Isolation Reagents (ThermoFisher Scientific, USA) as previously described. The 500ng of extracted RNA was reverse-transcribed into complementary DNA (cDNA) using the First-strand cDNA Synthesis Kit (K1072, Apexbio, USA) following the manufacturer’s protocol. qPCR was performed on the LightCycler 480 (Roche, Swiss) instrument by using 2X SYBR Green PCR Mix (K1070, Apexbio, USA). ΔΔCT was calculated to analyze the results of qPCR and data from each experiment were normalized to the expression of a control gene (GAPDH). The following primers were used:
ATP7A, forward 5′-TGACCCTAAACTACAGACTCCAA-3′ and reverse: 5′-CGCCGTAACAGTCAGAAACAA-3′; GAPDH, forward 5′-TCAACGGATTTGGTCGTATTGGGCG-3′ and reverse 5′-CTCGCTCCTGGAAGATGGTGATGGG-3′.
ATP7A, forward 5′-TGACCCTAAACTACAGACTCCAA-3′ and reverse: 5′-CGCCGTAACAGTCAGAAACAA-3′; GAPDH, forward 5′-TCAACGGATTTGGTCGTATTGGGCG-3′ and reverse 5′-CTCGCTCCTGGAAGATGGTGATGGG-3′.
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from tissues, cell lines, and CAFs using Total RNA Extraction Kits (Solarbio Science & Technology, Beijing, China) and synthesized to cDNA using the First-Strand cDNA Synthesis Kit (APExBIO Technology, Houston, TX, USA), followed by performing qRT-PCR with SYBR Green FAST Mastermix (Qiagen, Dusseldorf, Germany). The 2-ΔΔCT method was utilized to calculate relative expression. GAPDH and U6 were used as internal controls.
5
Transcriptional Analysis of TTHA1953 Disruption
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All reference and TTHA1953-disrupted strains were streaked onto TT plates prior to growth in liquid media. Single colonies from each streak were inoculated in TT media and grown at 70 °C, 180 RPM. For growth curves, starter cultures were grown overnight, and equivalent cell counts were inoculated into 30 ml cultures. Absorbance at 600 nm readings was performed by Synergy H1 Hybrid Reader (BioTek). For gene expression analysis, 1 ml samples of reference or TTHA1953-disrupted T. thermophilus HB8 cultures were collected and pelleted. RNA was isolated using a Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research). Resulting nucleic acid was treated with five units of DNaseI (Zymo Research) for 15 min at room temperature and then purified using an RNA Clean & Concentrator kit (Zymo Research). Complementary DNA (cDNA) libraries were generated using First-Strand cDNA synthesis kit (APExBIO) following the manufacturer’s protocol. Gene expression was quantified by qPCR using the qPCR primers listed in File S1 . qPCRs were assembled in a 10 μl volume containing 1× concentrate of iTaq Universal SYBR Green Supermix (Bio-Rad), 500 nM of each forward and reverse primer (File S1 ), and 2 μl diluted cDNA library. Expression levels were normalized to the endogenous pyruvate dehydrogenase E1 component gene (TTHA0185).
6
Reverse Transcription of DNase-Treated RNA
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DNase-treated RNA was reverse-transcribed using the First-Strand cDNA Synthesis Kit from APExBio (Boston, MA). Briefly, 1 μg of DNase-treated RNA was mixed with 1 μL Random Primers (50 μM) and 1 μL dNTP mixture (10 mM) and adjusted to 10 μL with the provided RNase-free water. To denature the RNA, the mixture was heated at 65°C for 5 min and then chilled on ice for 2 min. The reverse transcription reaction system was prepared by adding 4 μL First-Strand Buffer (5x), 1 μL RNase Inhibitor, Murine (40 U/μL), 1 μL Reverse Transcriptase (200 U/μL), and 4 μL RNase-free water to the denatured RNA mixture, for a total volume of 20 μL. The thermocycler conditions for cDNA synthesis were as follows: 25°C for 2 min, 42°C for 50 min, and 75°C for 15 min. The resulting cDNA templates were stored at -20°C until use.
7
Quantifying Fibroblast Gene Expression
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Total RNA was extracted from fibroblasts cultured on various matrices using a PureLink® RNA Mini Kit (Ambion, Grand Island, NY, USA). The yield of the extracted total RNA was determined by Nanodrop (Thermo Scientific NanoDrop 1000, Waltham, MA, USA). Reverse-transcription was carried out using an APExBIO First-Strand cDNA Synthesis kit (APExBIO Technology, Houston, TX, USA). RT-qPCR was performed using a Bio-Rad MyiQ single-color real-time PCR machine with PrimeTime Gene Expression master mix and primers (Integrated DNA Technologies, Coralville, IA, USA). Primers against the following genes were used in this study: COL1A1 (Hs.PT.58.15517795), COL3A1 (Hs.PT.58.4249241)), MMP2 L1 (Hs.PT.58.38701397) and ACTA2 (Hs405785835). GAPDH (Hs99999905_m1) was used as an endogenous reference. Data analysis was carried out using the 2−ΔΔCT method for relative quantification based on three replicates.
8
Investigating KLF3-AS1 and miR-338-3p in Osteosarcoma
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ScienCell provided the human normal osteoblast hFOB 1.19. OS cell lines HOS, Saos‐2, and SW1353 were obtained from American Type Culture Collection (ATCC). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and the Lipofectamine 3000 were sourced from Invitrogen. KLF3‐AS1‐siRNA (si‐lnc), MEF2C‐siRNA (si‐MEF2C) and its negative control (si‐NC), small hairpin targeting KLF3‐AS1 (sh‐lnc) and its sh‐NC, miR‐338‐3p mimic, mimic‐NC, miR‐338‐3p inhibitor (inhibitor), and inhibitor‐NC were all acquired from Sangon Biotech. The CCK‐8 kit and dimethyl sulfoxide (DMSO) used for cell viability assays were procured from Aladdin. Crystal violet and RIP assay‐related kit were from Sigma Aldrich. Primary antibodies (MEF2C, cat.no. ab211493; GAPDH, cat.no. ab8245; Bcl‐2, cat.no. ab194583; and Bax, cat.no. ab32503), and HRP‐conjugated secondary antibody (cat.no. ab6789) were bought from Abcam. The radioimmunoprecipitation assay (RIPA) buffer, enhanced chemiluminescence (ECL) detection kit, and PARIS™ Kit were from Thermo Fisher Scientific. The Total RNA Extraction Kit was acquired from Promega; the First‐Strand cDNA Synthesis Kit was from APExBIO Technology; and from Qiagen, we acquired a SYBR Green FAST Mastermix Kit (Dusseldorf, GER). Four‐ to five‐week‐old female BALB/c nude mice weighing 20–28 g, from Esebio, were used to establish tumor xenograft models.
9
Quantitative Analysis of Colonic Gene Expression
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Total RNA was isolated from the colonic tissues of control, DSS-induced colitis, and H-CeO2-PEG-treated DSS-induced colitis mice with TRIzol (Sigma). A First-Strand cDNA Synthesis Kit (APExBIO, Houston, USA) was used to carry out reverse transcription. qPCR analysis was carried out using the QIAGEN OneStep RT-PCR kit (QIAGEN, Germany) on an Applied Biosystems 7500 instrument (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The experiments were performed at least three times. Relative gene expression was calculated by the 2−ΔΔCT method.
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