Aortic wall biopsy specimens or cultured SMCs in RNAlater solution (Ambion, Austin, Tex) were used to isolate total RNA (0.5-1 μg) with Qiagen RNeasy kit (Valencia, Calif). Total RNA was reverse-transcribed with iScript cDNA Synthesis kit from Bio-Rad (Carlsbad, Calif). The gene-specific oligonucleotide sequences were as previously described.16 (link) Real-time RT-PCR was performed with the Stratagene Mx3005 machine and the SYBR Green JumpStart PCR kit (Sigma, St. Louis, Mo) as described by the manufacturer.
Sybr green jumpstart pcr kit
Manufactured by Merck Group
The SYBR Green JumpStart PCR kit is a reagent used for the amplification and detection of DNA sequences during the polymerase chain reaction (PCR) process. It contains all the necessary components, including SYBR Green I dye, for the efficient and accurate quantification of target DNA.
Automatically generated - may contain errors
Lab products found in correlation
Mx3005 machine, by Merck Group (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Qiashredder spin column, by Qiagen (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
3 protocols using sybr green jumpstart pcr kit
1
Aortic Smooth Muscle Cell RNA Isolation
2
Triglyceride Visualization and Gene Expression
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Accumulation of triglycerides was visualized by staining with Oil red O (Sigma-Aldrich), essentially as described previously [33] . Briefly, we prepared a stock solution of 0.5% (w/v) Oil red O in isopropanol. To achieve a working concentration, 6 mL of stock solution was mixed with 4 mL of water. RNA were isolated as described previously [4] (link) and converted to cDNA using SuperScript II RT (Invitrogen Life Technologies) and oligo dT24 primers. Quantitative RT-PCR was performed using the SYBR Green JumpStart PCR kit (Sigma-Aldrich, St. Louis, MO) according to the supplier's protocol. 36B4 and 18S RNAs were used as internal controls.
3
Quantitative Gene Expression Analysis
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For infection, cells were plated at a density of 3 × 105 cells per well in 6-well plates (for RT-qPCR analysis) or 6 × 105 cells per T25 flask (for RNA-seq analysis), and infected as described [3 (link)]. RNA was isolated with an RNA spin-column kit (5 PRIME) as recommended, except that cell lysates were passed through a QiaShredder spin column (Qiagen). RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and (dT)18 primer. RT-qPCR primers (listed in Supplementary Table s2 ) were designed based on gene-specific primer sequences in PrimerBank [50 (link), 51 (link)]. qPCR analysis was performed on an ABI 7500 with the SYBR Green JumpStart PCR kit (Sigma) with the inclusion of 5% DMSO, using the following program: one cycle of 95°C for 10 min pre-incubation, and 36 cycles of 95°C 10 s, 64°C 30 s. qPCR products were confirmed by melting curve analysis and gel electrophoresis.
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