Total RNA was extracted using TRIzol Reagent (Invitrogen), and reverse transcription was performed using the Advantage RT-for-PCR Kit (QIAGEN) according to the manufacturer’s instructions. For the real-time PCR analyses, aliquots of double-stranded cDNA were amplified using a SYBR Green PCR Kit (QIAGEN). The cycling parameters were as follows: 95 °C for 15 s, 55–60 °C for 15 s, and 72 °C for 15 s for 45 cycles. A melting curve analyses was then performed. The Ct was measured during the exponential amplification phase, and the amplification plots were analyzed using SDS 1.9.1 software (Applied Biosystems). For the cell lines, the relative expression levels (defined as the fold change) of the target genes were determined by the following equation: 2−ΔΔCt (ΔCt= ΔCttarget −ΔCtGAPDH; ΔΔCt = ΔCtexpressing vector −ΔCtcontrol vector). The expression level was normalized to the fold change that was detected in the corresponding control cells, which was defined as 1.0. For the clinical tissue samples, the fold change of the target gene was determined by the following equation: 2−ΔΔCt (ΔΔCt= ΔCttumor −ΔCtnontumor). This value was normalized to the average fold change in the normal colon epithelial tissues, which was defined as 1.0. All reactions were performed in duplicate. The primer sequences are listed in Table S6 .
Advantage rt for pcr kit
Manufactured by Qiagen
Sourced in Germany, United States
The Advantage RT-for-PCR Kit is a reagent kit designed for reverse transcription and subsequent PCR amplification of RNA targets. The kit contains all the necessary components for the two-step RT-PCR process, including a reverse transcriptase enzyme and a DNA polymerase for the PCR step. This product enables the conversion of RNA to cDNA and the amplification of the resulting cDNA for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr kit, by Qiagen (5 mentions)
Sds 1, by Thermo Fisher Scientific (3 mentions)
Rneasy plus mini kit 50 kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Merck Group (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Rotor gene real time dna amplification system, by Qiagen (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Titanium taq pcr kit, by Takara Bio (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
9 protocols using advantage rt for pcr kit
1
Real-Time PCR Expression Analysis
2
RNA Extraction and Analysis from Disc Cells
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RNA was extracted from L1-2, L2-3, and L3-4 marginal discs (noncartilaginous outgrowth) or hypertrophic CEP cells treated by LIG for 48 hours using 1 mL TRIzol reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's instruction. Cells/tissues were directly processed following RNA preparation employing the PURE Prep Kit protocol. One microgram of total RNA was reverse-transcribed using the Advantage RT-for-PCR Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Freshly transcribed cDNA (1 μL) was employed for qPCR using SYBR Green (Bio-Bad, Hercules, CA, USA) to monitor DNA synthesis with specific primers (Tables 1 and 2 ) designed by TaKaRa Biotechnology Co. Ltd. Gene expression was normalized to the housekeeping gene β-actin. PCR products were subjected to melting-curve analysis, and data were analyzed and quantified with the RotorGene 6.0 analysis software.
3
Quantitative Real-Time PCR Methodology
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Total RNA from cells was extracted using TRIzol reagent (Invitrogen, USA). One microgram of total RNA was reverse transcribed using the Advantage RT-for-PCR kit (Qiagen, Valencia, CA). Freshly transcribed cDNA was used for quantitative real-time PCR using SYBR Green (Bio-Rad, Hercules, CA). The primers for each gene were designed by online tool Primer3 (http://frodo.wi.mit.edu/ ) listed in supplementary Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/672312 . The PCR was carried out in a RotorGene real-time DNA amplification system (Corbett Research, Sydney, Australia) as described in our previous study [33 (link)].
4
Drebrin isoform expression in mouse lens
5
Quantifying mRNA Expression in HCC Cells
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Total RNA from HCC cells and samples was extracted using a RNeasy Plus Mini Kit (50) kit (Qiagen, Hilden, Germany) and then was reverse-transcribed using an Advantage RT-for-PCR Kit (Qiagen). RT-PCR was performed to amplify the target sequence using a SYBR Green PCR Kit (Qiagen). The relative mRNA expression was determined using the 2–ΔΔCt method and normalized to the control groups. The primers used for qRT-PCR were listed in Supplementary Table S7 .
6
qRT-PCR Analysis of BMSC Gene Expression
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BMSCs collected from ex vivo experiment were directly processed following RNA preparation employing the PURE Prep Kit protocol. One microgram of total RNA was reverse transcribed using the Advantage RT-for-PCR Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Freshly transcribed cDNA (1 μL) was employed for quantitative real-time PCR using SYBR Green (Bio-Bad, Hercules, CA) to monitor DNA synthesis with specific primers (Table 1 ) designed by NCBI/Primer-BLAST. Gene expression was normalized to the housekeeping gene β-actin. PCR products were subjected to melting-curve analysis, and the data were analyzed and quantified with the RotorGene 6.0 analysis software.
7
Quantitative Real-Time PCR Analysis
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The RNeasy Plus Mini Kit (50) kit (Qiagen, Hilden, Germany) was used to extract total RNA, which was then reverse transcribed with the Advantage RT-for-PCR Kit (Qiagen) in accordance with the manufacturer's protocols. The target sequence was amplified with real-time PCR with the SYBR Green PCR Kit (Qiagen). The cycling parameters used were 95 °C for 15 s, 55-60 °C for 15 s, and 72 °C for 15 s for 45 cycles. Melting curve analyses were performed, and Ct values were determined during the exponential amplification phase of real-time PCR. SDS 1.9.1 software (Applied Biosystems, Massachusetts, USA) was used to evaluate amplification plots. The 2-ΔΔCt method was used to determine relative fold changes in target gene expression in cell lines, which was normalized to expression levels in corresponding control cells (defined as 1.0). The equation used was 2-ΔΔCt (ΔCt = ΔCt target - ΔCt GAPDH; ΔΔCt = ΔCt expressing vector - ΔCt control vector). When calculating relative expression levels in surgically extracted CRC samples, relative fold changes in target gene expression were normalized to expression values in normal colon epithelial tissues (defined as 1.0) using the following equation: 2 -ΔΔCt (ΔΔCt = ΔCt tumor - ΔCt nontumor). All experiments were performed in duplicate. Table S5 lists all sequences of all primers used.
8
Quantitative RT-PCR Analysis of Gene Expression
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The RNeasy Plus Mini Kit (50) kit (Qiagen, Hilden, Germany) was used to extract total RNA, which was then reverse transcribed with the Advantage RT-for-PCR Kit (Qiagen) in accordance with the manufacturer’s protocols. The target sequence was amplified with real-time PCR with the SYBR Green PCR Kit (Qiagen). The cycling parameters used were 95 °C for 15 s, 55–60 °C for 15 s, and 72 °C for 15 s for 45 cycles. Melting curve analyses were performed, and Ct values were determined during the exponential amplification phase of real-time PCR. SDS 1.9.1 software (Applied Biosystems, Massachusetts, USA) was used to evaluate amplification plots. The 2–ΔΔCt method was used to determine relative fold changes in target gene expression in cell lines, which was normalized to expression levels in corresponding control cells (defined as 1.0). The equation used was 2–ΔΔCt (ΔCt = ΔCttarget – ΔCtGAPDH; ΔΔCt = ΔCtexpressing vector – ΔCtcontrol vector). When calculating relative expression levels in surgically extracted CRC samples, relative fold changes in target gene expression were normalized to expression values in normal colon epithelial tissues (defined as 1.0) using the following equation: 2 –ΔΔCt (ΔΔCt = ΔCttumor – ΔCtnontumor). All experiments were performed in duplicate. Supplementary Table S1 lists all sequences of all primers used.
9
Quantifying Gene Expression Changes
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An RNeasy Plus Mini Kit (50 reactions) (Qiagen, Hilden, Germany) was used to extract total RNA, which was then reverse transcribed with an Advantage RT-for-PCR Kit (Qiagen). The target sequence was amplified via qRT-PCR with a SYBR Green PCR Kit (Qiagen). The 2-ΔΔCt method was used to determine the relative fold changes (FCs) in target gene expression in the cell lines normalized to the levels in corresponding control cells (defined as 1.0). U6 small nuclear RNA and GAPDH were used as internal controls for miRNA and mRNA assays, respectively. In the 2-ΔΔCt equation, ΔCt=ΔCt target-ΔCt U6/GAPDH, and ΔΔCt=ΔCt expression vector-ΔCt control vector. All experiments were performed in duplicate. The PCR primers for miR-139-5p and U6 were purchased from RiBoBio (Guangzhou, China); the other PCR primers are listed in Supplementary Table 3 .
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