RNA was isolated using RNeasy Plus kits (Qiagen). SuperScript-III (Invitrogen) was used for reverse-transcription reactions. qPCR was performed with a LightCycler real-time PCR system using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics). DHRS9 primer sequences and cycling conditions are presented in Table 3 . DHRS9 signals were normalised against GAPDH mRNA expression. ALDH1A1, ALDH1A2, BCO1, BCO2, and CD1C were amplified using predesigned primer pairs (QuantiTect, Qiagen) per the manufacturer’s recommendations. PCR specificity was confirmed sequencing of amplicons (MWG Biotech).
Faststart dna master sybr green 1 kit
Manufactured by Roche
Sourced in Switzerland, Germany, United States, France
The FastStart DNA Master SYBR Green I kit is a reagent for real-time PCR analysis. It contains all the necessary components, including a DNA polymerase, SYBR Green I dye, and buffer, to perform quantitative PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler, by Roche (11 mentions)
Lightcycler 2, by Roche (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
High pure rna isolation kit, by Roche (6 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Lightcycler 2.0 instrument, by Roche (3 mentions)
Lightcycler 1, by Roche (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Cfx96 thermocycler, by Bio-Rad (2 mentions)
Lightcycler faststart dna master hybridization probe kit, by Roche (2 mentions)
Omniscript, by Qiagen (2 mentions)
Lightcycler system, by Roche (2 mentions)
Lightcycler 2.0 system, by Roche (2 mentions)
Lightcycler 480 system, by Roche (2 mentions)
Ex taq dna polymerase, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
53 protocols using faststart dna master sybr green 1 kit
1
Quantifying Gene Expression Levels
2
Quantifying Muscle Atrophy-Related Genes
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Using a small fraction (≈100 mg) of the biopsies, total RNA was extracted as described by Chomczynski and Sacchi.31 mRNA levels of E3 ligases (MuRF1, MAFbx, Nedd4, Fbxo30/MUSA1, Trim32, Hdm2, Ozz, and E4B), E2 Ub‐conjugating enzymes (UBE2A, UBE2B, UBE2D, UBE2E1, UBE2G1, UBE2J1, UBE2J2, UBE2L3, UBE2V1, UBE2V2, and UBE2N), proteasome subunits (PSMA1, PSMA3, PSMB1, PSMC1, PSMD2, PSMD4, PSMD7, and PSMD13), markers of apoptosis (Csp3, Csp9, Bax, and Bcl2), autophagy (CTPL and SQSTM1), and ATF4 pathway (4EBP1, ATF4, and CHOP) were determined by quantitative real‐time PCR (qRT‐PCR). Reverse transcription of total RNA was performed using the QuantiTect® Reverse Transcription kit (Qiagen®). qPCR was performed using the FastStart DNA Master SYBR Green I kit (Roche), according to the manufacturer's instructions using a CFX96 thermocycler (Bio‐Rad, Hercules, CA, USA). Calculations were made using the comparative ∆Ct method with YWHAZ, HPRT1, and 36B4 housekeeping genes. List of primers used is provided in Supporting Information, TableS1 .
3
Quantitative Real-Time PCR Protocol
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Total RNA from rat liver and FLC-5 cells was extracted with ISOGEN (Wako Pure Chem. Ind., Co., Ltd., Osaka, Japan) and the RNeasy Mini kit (Qiagen, Hilden, Germany), respectively. Gene expression in the RNA samples was evaluated by quantitative real-time PCR. First-strand cDNA was synthesized by reverse transcription (RT) using Omniscript (Qiagen). Subsequently, 2 µl of each RT reaction mixture was analyzed by LightCycler PCR (F. Hoffmann-La Roche Ltd. Diagnostics, Basel, Switzerland) using the LightCycler-FastStart DNA Master Hybridization Probe Kit or the FastStart DNA Master SYBR Green I Kit (Roche) according to the manufacturer’s protocols. The oligonucleotide primer sequences and accession numbers for target genes are shown in Table 1 . Expression of both human and rat α-TTP and β-actin, and rat CYP4F2 were analyzed using the LightCycler-FastStart DNA Master Hybridization Probe Kit, and the expression of rat glutathione peroxidase (GPx) and ABCA1 was analyzed using the FastStart DNA Master SYBR Green I Kit. Expression data for target genes were normalized to β-actin copy number to compensate for differences in RT efficacy among samples as previously described.(18 (link))
4
Real-Time PCR Primer Design and Quantification
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2.8.3.1 Design of primers. The primers for GAPDH (reference gene), Sox9, RunX2 and Osterix were designed with the aid of a program developed specifically for the elaboration of primers for the LightCycler (Roche Diagnostics GmbH, Germany). All the primers were verified relative to their specificity by means of analyzing the Melting curve, at all times using a positive and negative control.
2.8.3.2 RT-PCR reactions. The RT-PCR reactions were performed with the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany), using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany). The profile of reactions was determined in accordance with the protocol suggested by the equipment manufacturer. For each of the analyses, water was used as negative control, and the product of the reactions was quantified by using the program manufacturer’s own program (LightCycler Relative Quantification Software—Roche Diagnostics GmbH). The levels of the GAPDH gene expression were used as reference for normalization of the values (Table 1 ).
2.8.3.2 RT-PCR reactions. The RT-PCR reactions were performed with the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany), using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany). The profile of reactions was determined in accordance with the protocol suggested by the equipment manufacturer. For each of the analyses, water was used as negative control, and the product of the reactions was quantified by using the program manufacturer’s own program (LightCycler Relative Quantification Software—Roche Diagnostics GmbH). The levels of the GAPDH gene expression were used as reference for normalization of the values (
5
Quantification of Gene Expression by qPCR
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Total RNA was extracted using the TRIzol reagent (Invitrogen). Total RNA (2 μg) was reverse transcribed at 50°C for 2 min, followed by 60°C for 30 min. Quantitative PCR was performed using a FastStart DNA Master SYBR Green I kit and a LightCycler 480 detection system (Roche, Meylan, France), as specified by the manufacturer. The crossing point (Cp) was defined as the maximum of the second derivative of the fluorescence curve. Negative controls contained all elements of the reaction mixture, except the template DNA. For quantification, the relative mRNA expression of specific genes was obtained by the ΔΔCt method, using β-actin for normalization. The following gene-specific primers (5’→3’) were used: β-actin (forward, GAA ATC GTG CGT GAC ATC AAA G and reverse, TGT AGT TTC ATG GAT GCC ACA G), Foxp3 (forward, GGC CCT TCT CCA GGA CAG A and reverse GGC GCT GAT CAT TGG GTT GT), eGFP (forward, TGA ACC GCA TCG AGC TGA AGG G and reverse, TCC AGC AGG ATG TGA TCG C), RORγt (forward, TGT CCT GGG CTA CCC TAC TG and reverse, GTG CAG GAG TAG GCC ACA TT), and SOCS3 (forward, CGC CTC AAG ACC TTC AGC TC and reverse, CTG ATC CAG GAA CTC CCG AA).
6
Quantification of neuropeptide signaling
7
SFV E1 Gene Expression Quantification
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cDNA was produced from total RNA using Superscript II RNase H Reverse Transcriptase and amplified with primers targeting a 173 bp fragment of the E1 structural gene of SFV. The sequence of the primers was: 5′-CGC ATC ACC TTC TTT TGTG-3′ for the forward primer and 5′-CCA GAC CAC CCG AGA TTT T-3′ for the reverse primer. Real-time PCR was performed using FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland). An initial denaturation step at 95 °C for 10 min was followed by 40 cycles of amplification. Each cycle comprised denaturation at 94 °C for 10 s, annealing at 62 °C for 5 sec and extension at 72 °C for 10 sec. The Tm of the 173 bp product was approximately 88.5 °C. For the quantitative PCR standards, an in vitro transcript from the pGEM1-SFV cDNA plasmid containing the structural genes of SFV was transcribed using a Promega RiboMax kit (Southampton, UK). Serial dilutions of the plasmid were made to produce a standard curve.
8
Quantitative Gene Expression Analysis in Liver
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Total RNA was extracted from the livers and hepatocytes using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, US) according to the manufacturer’s specifications. Reverse transcription-PCR and real-time quantitative PCR analysis were performed. Briefly, reverse transcription of total RNA was performed using an avian myeloblastosis virus RT with a first-strand complementary DNA synthesis kit for reverse transcription-PCR. Aliquots of the reverse transcription reactions were then submitted in duplicate to online quantitative PCR with the LightCycler® 480 Real-Time PCR system (Roche Applied Science) with SYBR green using the FastStart DNA-Master SYBR Green I kit (Roche Applied Science). Relative abundance of mRNA was calculated after normalization to 18S ribosomal RNA. Specific primers were synthesised commercially (Shanghai Sangon Biological Engineering Technology and Services Company Ltd, Shanghai, China). Sequences for the primers used in this study are shown in Table S1 .
9
Quantitative RT-PCR analysis of renal gene expression
10
Real-time PCR Analysis of EMT Markers
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Real-time PCR was performed with the Light Cycler 2.0 (Roche) using the Fast Start DNA Master SYBR Green I Kit (Roche). For verification of the correct amplification product, PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. The sequences of the primers were designed as follows: for β-actin, 5′-GACTATGACTTAGTTGCGTTA-3′ and 5′-GCCTTCATACATCTCAAGTTG-3′, for snail, 5′-GGCTCCTTCGTCCTTCT-3′ and 5′-GGCTGAGGTATTCCTTGTT-3′, for twist1, 5′-CGGGAGTCCGCAGTCTTA-3′ and 5′-CTGGTAGAGGAAGTCGATGT-3′, for c-myc, 5′- GCTTTATCTAACTCGCTGTAGTAAT-3′ and 5′- GCTGCTATGGGCAAAGTTTC-3′. Primers of MMP7 (P310408) and MM9 (P323207) were purchased from Bioneer. PCR was conducted at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 seconds, 60 °C for 5 seconds, and 72 °C for 7 seconds. Melt curve analysis was performed to confirm that a single product was present. Negative controls without template were included in each run. Data were analyzed using Light Cycler software version 4.0 (Roche, Switzerland). The 2ΔΔCt method was used for analysis of relative gene expression.
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