Quantitative reverse transcriptase-PCR was performed as previously described.16 (link) RNA was isolated from the cell lines at each passage using TRIZOL (Invitrogen). Complementary DNA was synthesized from 1 mg of total RNA using oligo dT18 primers and Superscript reverse transcriptase (Bioneer, Daejeon, Korea) in a final volume of 20 μl. For standard PCR, 1 μl of first-strand complementary DNA was used as a template for PCR amplification with Taq DNA polymerase (Fermentas, Grand Island, NY, USA). Quantitative reverse transcriptase-PCR reactions were performed using SYBR Green JumpStart Taq ReadyMix (Takara, Shiga, Japan) with an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer). Relative quantification was achieved by normalization to endogenous β-actin. The primers used are shown in Table 1 .
Superscript reverse transcriptase
Manufactured by Bioneer
Superscript reverse transcriptase is an enzyme used in the reverse transcription process, which involves converting single-stranded RNA into complementary DNA (cDNA). This enzyme plays a crucial role in the conversion of genetic information from RNA to DNA, a fundamental step in various molecular biology and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Ugenius 3 gel documentation system, by Syngene (5 mentions)
Oligo dt18, by Bioneer (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nucleozol reagent, by Macherey-Nagel (2 mentions)
Sybr green jumpstart taq readymix, by Takara Bio (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Exicycler 96 real time quantitative thermal block, by Bioneer (1 mentions)
Taq dna polymerase, by Cosmogenetech (1 mentions)
8 protocols using superscript reverse transcriptase
1
Quantitative RT-PCR Protocol for Gene Expression Analysis
2
Quantification of BAFF, VCAM1 and Integrin β1 Expression
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Total RNA was extracted from MH7A cells using Nucleozol® (BMS). cDNA was synthesized from 1ug of total RNA using oligo-dT18 primers (Macrogen, Seoul, Korea) and superscript reverse transcriptase (Bioneer, Daejeon, Korea) in a total volume of 21 uL. For standard PCR, 1 µL of the first-strand cDNA product was then used as a template for PCR amplification with Taq DNA polymerase (Cosmo Genetech, Seoul, Korea). PCR amplification was performed using 10 pmol of specific primers specific for human BAFF (forward; aat tca gag gaa ggt cc, reverse; atg tga cat ctc cat cca gt), VCAM1(forward; gga acg aac act ctt acc t, reverse; gca act gaa cac ttg act g), integrin β1 (forward; cag cag ttg gtt ttg cga tt, reverse; atg cgc tgt ttt cca aca ag) and β-actin (forward;gtc acc aac tgg gac gac at, reverse; gca cag cct gga tag caa cg) with 36 thermocycles (95 °C for 40 s, 57 °C for 30 s and 72 °C for 60 s). PCR products were detected by agarose gel electrophoresis.
3
Total RNA Extraction and RT-PCR Analysis
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Total RNA was extracted by using TRizol reagent (Invitrogen, Calsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT18, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20 μl. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 25 ~ 35 thermocycles for 30 sec at 95 °C, 30 sec at 55 °C, and 60 sec at 72 °C using human (h) or mouse (m) oligonucleotide primers specific for hTB4 (sense: ACA AAC CCG ATA TGG CTG AG; anti-sense: CCT CCA AGG AAG AGA CTG AA), mTB4 (sense: ATA TGG CTG AGA TCG AGA AA; anti-sense: GCT TGC TTC TCT TGT TCA AT), hGAPDH (sense: GAA GGT GAA GGT CGG AGT C; anti-sense: GAA GAT GGT GAT GGG ATT TC) and mGAPDH (sense: TCC ACC ACC CTG TTG CTG TA; anti-sense: ACC ACA GTC CAT GCC ATC AC). Amplified PCR products were separated by 1.0 ~ 1.5% agarose gel electrophoresis and detected on Ugenius 3® gel documentation system (Syngene, Cambridge, United Kingdom).
4
RNA Extraction, cDNA Synthesis, and Gene Expression Analysis
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Total RNA was extracted by using NucleoZOL reagent (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT18, and superscript reverse transcriptase (Bioneer, Daejeon, Republic of Korea) in a final volume of 20 μL. For standard PCR, 1 μL of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 30~35 thermocycles for 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C using human oligonucleotide primers specific for NPHP3 (sense: 5′-AGC GAA ATA CCA AGC AAT GG-3′; anti-sense: 5′-TGG AAG GTT CAC TTC CCA AG-3′), HIF-1α (sense: 5′-CTC AAA GTC GGA CAG CCT CA-3′; anti-sense: 5′GAT TGC CCC AGC AGT CTA CA-3′) and actin (sense: 5′-GTC ACC AAC TGG GAC GAC AT-3′; anti-sense: 5′-GCA CAG CCT GGA TAG CAA CG-3′). Amplified PCR products were separated by 1.0–2.0% agarose gel electrophoresis and detected on Ugenius 3® gel documentation system (Syngene, Cambridge, UK).
5
Reverse Transcription-PCR for Gene Expression
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Total RNA was extracted by using TRizol reagent (Invitrogen, Calsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT18, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20ul. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 25~35 thermocycles for 30 sec at 95 °C, 30 sec at 55 °C, and 60 sec at 72 °C using human (h) oligonucleotide primers specific for hTβ4 (sense: ACA AAC CCG ATA TGG CTG AG; anti-sense: CCT CCA AGG AAG AGA CTG AA), hNPHP3 (sense: AGC GAA ATA CCA AGC AAT GG; anti-sense: TGG AAG GTT CAC TTC CCA AG), hGAPDH (sense: GAA GGT GAA GGT CGG AGT C; anti-sense: GAA GAT GGT GAT GGG ATT TC). Amplified PCR products were separated by 1.0~1.5% agarose gel electrophoresis and detected on Ugenius 3® gel documentation system (Syngene, Cambridge, United Kingdom).
6
RNA Extraction and Gene Expression Analysis
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Total RNA was extracted by using TRizol reagent (Invitrogen, Calsbad, CA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT18, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20 μl. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 30 ~ 35 thermocycles for 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C using oligonucleotide primers specific for human TB4 (sense: ACA AAC CCG ATA TGG CTG AG; anti-sense: CCT CCA AGG AAG AGA CTG AA), GAPDH (sense: GAA GGT GAA GGT CGG AGT C; anti-sense: GAA GAT GGT GAT GGG ATT TC) and actin (sense: GTC ACC AAC TGG GAC GAC AT; anti-sense: GCA CAG CCT GGA TAG CAA CG). Amplified PCR products were separated by 1.0–2.0% agarose gel electrophoresis and detected on Ugenius 3® gel documentation system (Syngene, Cambridge, United Kingdom)17 (link).
7
RNA Extraction and Gene Expression Analysis
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Total of RNA was extracted from WiL2-NS cells by Nucleozol (BMS). 1 µg of total RNA was used for synthesizing cDNA. With using Oligo (dT) 18 mer primer (macrogen) and superscript reverse transcriptase (Bioneer) were added with follows: PCR amplification was performed to detect hCXCR4 (using primer forward; 5′-CAG TGA GGC AGA TGA CAG AT-3′, reverse; 5′-CAG GAC AGG ATG ACA ATA CCA-3′), and β-actin (using primer forward; 5′-GTC ACC AAC TGG GAC GAC AT-3′, reverse; 5′-GCA CAG CCT GGA TAG CAA CG-3′).
8
ACE2 Expression Analysis by RT-PCR
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Total RNA was extracted by using NucleoZol reagent (MACHEREY-NAGEL GmbH & Co., Duren, Germany). Complementary DNA (cDNA) was synthesized from 1 μg of isolated total RNA, oligo-dT18, and superscript reverse transcriptase (Bioneer, Daejeon, Rep. of Korea) in a final volume of 20 μl. For standard PCR, 1 μl of template cDNA was amplified with Taq DNA polymerase. PCR amplification was performed with 25 ~ 35 thermocycles for 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C using human (h) oligonucleotide primers specific for ACE2 (sense: 5′-cat tgg agc aag tgt tgg atc tt-3′; anti-sense: 5′-gag cta atg cat gcc att ctc a-3′) [26] , and β-actin (sense: 5′-gtc acc aac tgg gac gac at-3; anti-sense: 5′-gca cag cct gga tag caa cg-3′). Amplified PCR products were separated by 1.0 ~ 1.5% agarose gel electrophoresis and detected on Ugenius 3® gel documentation system (Syngene, Cambridge, United Kingdom) [23] (link).
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