The expression of IL13Rα2 in U87MG cells growing as an adherent culture or as neurospheres was characterized by qRT-PCR. RNA was isolated from glioma cells using RNeasy plus kit (Qiagen, Boston, MA) and was reverse-transcribed using iScript cDNA conversion kit (Biorad, CA, USA). qRT-PCR reaction was carried out using SYBR green qPCR kit (Biorad, CA, USA) using following primers: GAPDH forward primer: 5′-GGTCGGAGTCAACGGATTTGG-3′; GAPDH reverse primer: 5′-CATGGGTGGAATCATATTGGAAC-3′; IL13Rα2 forward primer: 5′-TTGGGACCTATTCCAGCAAGGTGT-3′; IL13Rα2 reverse primer: 5′-CACTCCACTCACTCCAAATCCCGT-3′. For relative quantification of IL13Rα2 expression, all IL13Rα2 values were normalized to the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) expression values. Data analysis was performed using the 2−ΔΔCT method46 (link).
Sybr green qpcr kit
Manufactured by Bio-Rad
Sourced in United States
The SYBR green qPCR kit is a real-time PCR reagent used for quantitative analysis of DNA and RNA samples. The kit contains SYBR green, a fluorescent dye that binds to double-stranded DNA, allowing for the detection and quantification of target sequences during the PCR amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Amv reverse transcriptase, by Promega (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Iscript cdna conversion kit, by Bio-Rad (2 mentions)
Rneasy plus kit, by Qiagen (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Cfx96 touch, by Bio-Rad (2 mentions)
Myiq real time pcr detection system, by Bio-Rad (2 mentions)
First strand cdna synthesis kit, by Bio-Rad (2 mentions)
Quantstudio 3, by Bio-Rad (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
M6a antibody, by Merck Group (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Rnasin, by Promega (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcriptase system, by Promega (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
29 protocols using sybr green qpcr kit
1
Quantifying IL13Rα2 Expression in Glioma Cells
2
Quantitative Transcriptome Analysis
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Total RNA was harvested using TRIzol according to the manufacturer’s protocol. cDNAs (complementary DNA) were synthesized from 1 μg total RNA using avian myeloblastosis virus (AMV) reverse transcriptase (Promega) and used directly for qPCR analysis with the SYBR green qPCR kit (Bio-Rad). Ct values (cycle thresholds) signals obtained by qPCR were normalized to those for 18S unless otherwise noted.
3
Methylation-Specific RNA Immunoprecipitation and Analysis
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HEK293T or iSLK cells were transfected as indicated and used for meRIP (methylated [m6A] RNA immunoprecipitation); then, total RNA was extracted using TRIzol. Pulldowns were performed using protein G Dynabeads (Invitrogen) with 10 μg m6A antibody (Sigma-Aldrich) and 100 μg RNA in meRIP buffer (50 mM Tris⋅HCl at 7.4 pH,150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, Millipore H2O) and 1 μL RNAsin (RNAse inhibitor - Promega) per sample overnight at 4 °C. After extensive washing, samples are eluted in meRIP buffer containing 6.7 mM sodium salt for 30 min at 4 °C. cDNAs were then obtained from 1 μg total RNA using AMV reverse transcriptase (Promega) and used directly for qPCR analysis with the SYBR green qPCR kit (Bio-Rad).
4
Quantification of Osteogenic Gene Expression
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RNA was collected from flushed long bones of mice. Total RNA was isolated after homogenization using the Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. RNA (5 μg) was subjected to reverse transcription using the SuperScript III first‐strand synthesis kit (Invitrogen). Gene expression was determined by real‐time PCR using the CFX96 real‐time PCR detection system (Bio‐Rad Laboratories, Hercules, CA, USA) and the SYBR green qPCR kit (Bio‐Rad) using primers for Runx2,(10 ) Osterix (Sp7),(10 ) collagen Iα1 (ColIa1),(11 ) osteopontin (Spp1),(10 ) osteocalcin (Bglap),(12 ) sclerostin (Sost),(13 ) bone sialoprotein (Ibsp),(10 ) alkaline phosphatase (Alpl),(10 ) matrix metalloproteinase 13 (Mmp13),(14 ) osteoprotegerin (Tnfrsf11b),(10 ) and RANKL (Tnfsf11)(10 ) according to previously published protocols. Primer sequences are provided in Supplemental Table S1 . Target gene expressions were normalized to β‐actin, and the relative changes in mRNA levels were assessed by the comparative CT method.
5
Quantitative Analysis of Breast Cancer Transcripts
6
Quantitative Analysis of CRM1 Expression
7
Quantifying Osteoblast Gene Expression
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Total RNA was isolated from primary osteoblasts (obtained by serial collagenase digestion on flushed long bones) and primary tumors after homogenization using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA (1.5 μg) was subjected to reverse transcription using the iScript RT kit (Bio-Rad), and gene expression was determined by real-time PCR using the CFX96 real-time PCR detection system (Bio-Rad) with gene-specific primers (17 (link)) and the SYBR green qPCR kit (Bio-Rad). Target gene expressions were normalized to β-actin and the relative changes in mRNA levels were assessed by the comparative Ct method (67 (link)).
8
RNA Extraction and qPCR Analysis
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Total RNA was extracted and purified from cells by RNeasy Plus Universal Mini Kit (QIAGEN, Valencia, CA) and reverse transcribed to cDNA in a 20 μL volume. Quantitative real-time PCR analysis was performed using SYBR Green q-PCR kit (Bio-Rad, Hercules, CA). The level of the different mRNAs was normalized to GAPDH. The primer sequences refered to published paper [7 (link)].
9
Yeast Total RNA Extraction and RT-qPCR Analysis
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Total RNA was purified from yeast cultures using phenol extraction (Köhrer and Domdey, 1991 (link)). RNA samples were resuspended in RNase-free water and quantified by spectrophotometry. cDNA was synthesized by reverse transcription using random hexamer DNA primers (Thermo Fisher Scientific), SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and 1 μg total RNA as described previously (Kimmig et al., 2012 (link)). 1% of the cDNAs was employed for qPCR reactions using SYBR green qPCR kit (Bio-Rad). qPCR was performed in triplicates using CFX96 Touch Real-Time PCR Detection System (Bio-rad). qPCR primers are listed in Table 2 . mRNA levels were normalized to NDA2 mRNA in S. pombe.
10
Quantitative Real-Time PCR Protocol for Gene Expression Analysis
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Primers for various genes identified after the subtraction hybridization (Table I ) were synthesized. Specific gene primer pairs for each isolated gene were designed to perform qPCR using SYBR Green qPCR kit (BioRad Laboratories, Inc.) according to the manufacturer's protocol. The primer sequences for ZFPL1 mRNA were as follows: sense, 5′-agg-ccc-agt-gaa-aga -gat-ca-3′ and antisense, 5′-aag-tgc-ccc-aag-aga-aag-gt-3′. The internal reference gene was GAPDH. The primer sequences were as follows: sense, 5′-acg-ccg-cat-ctt-ctt-gtg-c-3′ and anti-sense-5′-aca-gcc-gca-tct-tct-tgt-gc-3′.
Total RNA from frozen prostate tissue specimens or PC cell lines was extracted using TRIzol® (Ambion; Thermo Fisher Scientific, Inc.). The extracted RNA was verified for integrity before reverse transcription. A total of 1 µg RNA/sample was reverse transcribed, and 25 ng cDNA was used for RT-qPCR. RT-qPCR was performed using Perfecta Fast MIX kit (Quantabio). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec followed by 35 cycles of amplification as follows: Initial denaturation phase at 95°C for 15 sec, annealing phase at 60°C for 30 sec and extension phase at 72°C for 10 sec. Relative expression was calculated using the comparative cycle threshold method (2−ΔΔCq), and the results were expressed relative to a normal tissue or a parental cell line (31 (link),32 (link)).
Total RNA from frozen prostate tissue specimens or PC cell lines was extracted using TRIzol® (Ambion; Thermo Fisher Scientific, Inc.). The extracted RNA was verified for integrity before reverse transcription. A total of 1 µg RNA/sample was reverse transcribed, and 25 ng cDNA was used for RT-qPCR. RT-qPCR was performed using Perfecta Fast MIX kit (Quantabio). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec followed by 35 cycles of amplification as follows: Initial denaturation phase at 95°C for 15 sec, annealing phase at 60°C for 30 sec and extension phase at 72°C for 10 sec. Relative expression was calculated using the comparative cycle threshold method (2−ΔΔCq), and the results were expressed relative to a normal tissue or a parental cell line (31 (link),32 (link)).
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