Testis total RNAs were isolated as reported previously (Fan et al. 2006b (link)). RT-PCR was conducted by the random primer method as described in the RT-for-PCR kit (Clontech). The primers for β-actin were (5’-GTGGGCCGCTCTAGGCACCAA-3′ and 5’-CTCTTTGATGTC ACGCACGATTTC-3′). The primers FP1 (5′-CACCCAGCTCATCTACTTAGC-3′, located on exon 1) and RP2 (5′-GAGCAGGGTTGTTCCTGTATAG-3′, located on exon 4) were used to produce an amplicon of 700 bp to identify the long isoform Ptchd3b (Fan et al. 2007 (link)). The primers for Ptch1 were forward 5’-TATGCTCGCTCTGGAGCACA-3′ and reverse 5’-TCTGTGGCTTCCACAATCAC-3′. The primers for Ptch2 were forward 5′- CAATGATGACTGTGGAGCTC-3′ and reverse 5′- AGAACCAGCAAGCATGAGCA-3′. The primers for Smoothened were forward 5′- ACCTCAATGAACCCTCAGCT-3′ and reverse 5′- CTCAGCCTCCATTAGGTTAG-3′. The primers for Gli1 were forward 5′- TCGGAAGTCCTATTCACGCCTTGA-3′ and reverse 5′- CCATGCACTGTCTTCACGTGTTTG-3′. The primers for Wsb2 were forward 5’ TAAGCACGGTAAGCAGATCCAGGT-3′ and reverse 5′- CCAGATCCTGAGCAGCCTGTCATC-3′. PCR parameters: 94 °C for 2 min, 1 cycle; 94 °C for 20 s, 58 °C for 20 s, 72 °C for 90 s, 30 cycles; followed by a 6-min extension at 72 °C. All PCR products were analyzed on 1% agarose gel.
Rt for pcr kit
Manufactured by Takara Bio
Sourced in Japan
The RT-for-PCR kit is a laboratory tool designed for the reverse transcription (RT) step of the polymerase chain reaction (PCR) process. It enables the conversion of RNA into complementary DNA (cDNA), which can then be amplified and analyzed using PCR techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Cfx system, by Bio-Rad (3 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler480 384 well pcr system, by Roche (1 mentions)
Lightcycler probe design software, by Roche (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Sybr green 2, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Stepone plus thermal cycler, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyser rna 6000 nano kit, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr mix, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
21 protocols using rt for pcr kit
1
Testis RNA Isolation and RT-PCR Analysis
2
Quantitative Analysis of PTK7 mRNA
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Total RNA was extracted from clinical samples using the TRIzol reagent (Invitrogen, Waltham, MA, USA) and was reversed transcribe to cDNA following the vendor’s protocols (RT-for-PCR kit, Clontech Laboratories, Mountain View, CA, USA). The resultant cDNA corresponding to the PTK7 mRNA was measured using quantitative real-time PCR (qPCR) using a SYBR Green PCR Kit (Applied Biosystems, Foster City, CA, USA) and a LightCycler480 384-well PCR system (Roche Diagnostics, Basel, Switzerland). The ACTB gene (encoding β-actin) comprised the internal control for PTK7. PTK7 primers were 5′ACACTTCGTTGCCACATTGAT-3′ (forward) and 5′CAGCAGGAATACAGCCCAC-3′ (reverse). ACTB primers were 5′CATTAAGGAGAAGCTGTGCT-3′ (forward) and 5′GTTGAAGGTAGTTTCGTGGA-3′ (reverse). The relative expression of the gene in each sample was averaged and compared using the cycle threshold (Ct) method. The 2–Ct method was used to calculate the fold changes in mRNA levels.
3
Quantitative PCR Analysis of Rat Genes
4
RNA Extraction and qPCR Analysis
5
Quantifying Gene Expression in Lung Cancer
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Human lung cancer tissues or indicated cells were subjected to total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocols. Then, 1 ug of the RNA was reversely transcribed into cDNA using RT-for-PCR kit (Clontech, Takara, Japan). Quantification of mRNA level was performed using SYBR Green II (Takara, Japan). The primer sequence was as follow: CCT3 forward, 5’-TCAGTCGGTGGTCATCTTTGG-3’ and reverse, 5’-CCTCCAGGTATCTTTTCCACTCT-3’; Ki-67 forward, 5’-AGAAGAAGTGGTGCTTCGGAA-3’ and reverse, 5’-AGTTTGCGTGGCCTGTACTAA-3’; PCNA forward, 5’-CCTGCTGGGATATTAGCTCCA-3’ and reverse, 5’-CAGCGGTAGGTGTCGAAGC-3’; Cyclin D1 forward, 5’-CAATGACCCCGCACGATTTC-3’ and reverse, 5’-CATGGAGGGCGGATTGGAA-3’; GPX4 forward, 5’-GAGGCAAGACCGAAGTAAACTAC-3’ and reverse, 5’-CCGAACTGGTTACACGGGAA-3’; GAPDH forward, 5’-TGTGGGCATCAATGGATTTGG-3’ and reverse, 5’-ACACCATGTATTCCGGGTCAAT-3’. The 2−ΔΔCt method was applied to determine mRNA expression and GAPDH acted as the internal control.
6
RNA Isolation and RT-qPCR Analysis
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Total RNA was isolated from cell lines and tissues by Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Agarose gel electrophoresis and spectrophotometric analysis (A260: 280 nm ratio) were used to evaluate RNA quality and quantity. RT-for-PCR kit (Clontech, Palo Alto, CA) with random priming as recommended in the protocol provided. All primers and reaction conditions are listed in Additional file 1 : Table S1. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. For RT-PCR, the PCR products were separated in 2% agarose gel in electrophoresis and visualized with ethidium bromide staining, and quantified using an image analysis system (Gel work-2ID). Real time PCR reactions were performed by the Stepone Plus Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and SYBR green PCR Master Mix (Life Technology, Foster City, CA, USA). The expression levels of target genes were normalized with GAPDH using the 2-△△CT method [24 (link)]. The reaction was repeated in triplicate with each of the samples for quality control.
7
RNA Extraction and qPCR Analysis
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RNA was extracted according to manufacturers instructions with the RNeasy kit from Qiagen (Valencia, CA). cDNA was prepared with the RT-for-PCR kit from Clontech (Mountain View, CA) or Quantitect Reverse Transcription kit (Qiagen). QPCR was performed with the SybrGreen Master Mix (Applied Biosciences) in a 7900HT cycler (Applied Biosciences). Cyclophilin or TBP was used for normalization. Sequences of the primers are available upon request.
8
RNA Extraction and cDNA Synthesis
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RNA was prepared from FP tissue of individual mice as described previously [15] (link). cDNA was generated from 0.5 µg RNA using random hexamers and an RT-for-PCR kit (Clontech, Palo Alto, CA) at 42°C for 1 hr in a thermocycler (9600, Perkin-Elmer Corp., Norwalk, CT). Controls for DNA contamination were prepared from RNA samples using the reverse transcription reagents minus the reverse transcriptase. All sample and control preparations were aliquoted and stored at −70°C.
9
RNA Extraction and cDNA Synthesis
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RNA was extracted from RNAlater-preserved normal skin and CMCT samples using either TRIzol (Life Technologies) or the RNAeasy kit (QIAGEN). RNA integrity was evaluated by microfluidic electrophoresis (Agilent 2100 Bioanalyser RNA 6000 Nano Kit). cDNA was synthesised using the RT-for-PCR kit (Clontech).
10
Investigating KLF5 and STK24 Regulation
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A549 and H1299 cells were obtained from American Type Culture Collection (Manassas, USA). Dulbecco's modified eagle (DMEM), 1640 cell culture medium, and antibiotics were from Corning. Fetal bovine serum (FBS) was purchased from Gibco (California, USA). Antibodies against KLF5, STK24, β-actin, and all of the secondary antibodies were from Proteintech (Wuhan, China). siRNAs against negative control, STK24, and KLF5 were obtained from GenePharma (Shanghai, China). TRIzol reagent was from Invitrogen (Carlsbad, USA). The RT-for-PCR kit was from Clontech. SYBR Green qPCR mix was from Takara (Japan). Protease and phosphatase inhibitors were purchased from Roche (Basel, Switzerland).
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