Chondrocytes and synovial cells were harvested at the 3rd, 5th, and 8th days of culture, and RNA was extracted using TRIzol (Invitrogen, Penrose, Auckland, NZ) and quantified with a spectrophotometer. cDNA of the extracted mRNA was synthesized with a SuperSCRIPT First-Strand Synthesis RT-PCR kit (Invitrogen, Penrose, Auckland, NZ) by combining RNA, oligo (dT), and deoxynucleotide triphosphates (dNTPs) and incubating for 5 min at 65℃, then adding 10X RT Buffer, 25 Mm MgCl2, 0.1 M dithiothreitol (DTT), RNaseOUT, and SuperScript and incubating for 50 min at 50℃ and for 5 min at 85℃, then treating with RNase H for min at 37℃. Sequences of the forward and reverse primers of VEGF-A and PEDF are given in Table 1 . Five microliters of the synthesized cDNA, 1 µL of 10 pmol forward and reverse primers, 10 µL of iQ SYBER Green Supermix (Bio-Rad, Hercules, CA, USA), and 3 µL of nuclease-free water were mixed and subjected to 55 cycles of PCR (15 sec at 95℃, 15 sec at 56℃, and 15 sec at 72℃). The results were analyzed with Bio-Rad IQ5 software. The concentrations of mRNA of VEGF-A and PEDF were calculated with reference to a standard curve and compared to the amount of β-actin.
Superscript first strand synthesis rt pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in New Zealand
The SuperSCRIPT First-Strand Synthesis RT-PCR kit is a reagent kit designed for reverse transcription and subsequent PCR amplification of RNA samples. The kit provides essential components for the conversion of RNA to cDNA and the amplification of the generated cDNA.
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Lab products found in correlation
4 protocols using superscript first strand synthesis rt pcr kit
1
Chondrocyte and Synovial Cell VEGF-A and PEDF Quantification
2
Cloning mVipr1 into pAAV-hDLX-T2A-eGFP
3
Quantifying HERVK3-1 Expression in Glioma Cells
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RNA was isolated from NHA, A172, GBM28, and GBM43 using Trizol extraction with chloroform as previously described44 (link). RNA was purified using DNase and quantified using NanoDrop 2000 (ThermoFisher Scientific) for a 260/280 value ~ 2.0, and reverse transcribed using the SuperScript First-Strand Synthesis RT-PCR kit (Thermo Fisher Scientific). Polymerase chain reactions were then used to amplify HML-6 transcripts on the cDNA using primers (10 uM) which target the protein-coding region of HML-6 (ERVK3-1). Fast SybR green master mix was used for the PCR with the following cycling conditions: 95 °C for 20 s, [95 °C for 3 s, 60 °C for 30 s] repeated for 40 cycles followed by 95 °C for 20 s [95 °C for 1 s, 60 °C for 20 s] for 40 cycles. Both primers targeted the env region of the HERVK3-1 downstream of LTR13. Ct values were normalized to expression of NHA using the delta-delta Ct method45 (link). Reverse transcriptase negative controls were included and did not amplify.
4
Transcription of HML-2 env in Cell Lines
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RNA was isolated from iPSC, NSC, neuronal, and AT/RT cell lines with a Qiagen RNeasy kit (Qiagen, 74104) and reverse transcribed with the SuperScript First-Strand Synthesis RT-PCR kit (ThermoFisher Scientific, 11904018). Polymerase chain reactions were used to amplify HML-2 envelope transcripts with primers which target the full length env transcript (Table 1 ). Q5 high fidelity 2X master mix was used for the PCR with the following cycling conditions: 98 °C for 90 s, [98 °C 10 s, 55 °C 20 s, 72 °C 4 min] repeat 34 times, final extension at 72 °C for 6 min, 4 °C forever. The PCR primers were used to amplify the full length env gene as following: forward primer 5’- cccactagacatttgaagttctaca-3’, and reverse primer 5’- ggagtctcctatgtctacttcttt-3’. One primer aligned to the 3’ LTR of the HML-2 element and the other primer was positioned 5’ (upstream) of the start of the env gene. For the AT/RT cells, we cloned products into Topo-TA vectors (ThermoFisher Scientific, K203001) and sent them for Sanger sequencing to determine which env loci were actively transcribed in the samples. For the iPSCs, NSCs, and neurons, PCR products were run in a 2% agarose gel with GelStar Nucleic Acid Gel stain (Lonza, 50535) and the gel was imaged using Flourchem Protein Simple imager.
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