Total RNA was isolated from OVCAR3, RMG1, and SKOV3 cells using ISOGEN (Nippongene, Japan). One microgram of total RNA was reverse-transcribed to cDNA by SuperScript III (Invitrogen, Carlsbad, CA, USA) in a final volume of 20 μL. Then, 0.067 μL of cDNA was used to perform quantitative PCR reactions with KAPA SYBR® FAST ABI Prism® (Kapa Biosystems, Foster City, CA, USA) on Step One Plus (Applied Biosystems, Foster City, CA, USA). GAPDH was used as the normalization control. The primers are shown in Supplementary Table S2 .
Kapa sybr fast abi prism
Manufactured by Roche
Sourced in United States
KAPA SYBR FAST ABI Prism is a laboratory equipment product designed for real-time PCR (polymerase chain reaction) analysis. It is a master mix formulation that enables fast and sensitive qPCR (quantitative PCR) reactions on Applied Biosystems (ABI) real-time PCR instruments.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Universal cdna synthesis kit 2, by Qiagen (1 mentions)
Exilent sybr green, by Qiagen (1 mentions)
Abi7900 apparatus, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dnase treatment, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rna stabilization reagent, by Qiagen (1 mentions)
Kapa sybr fast qpcr master mix, by Roche (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
7 protocols using kapa sybr fast abi prism
1
Quantifying Gene Expression in Ovarian Cancer
2
Quantification of miR-146a and TNF-α Expression
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Total RNA was isolated from individual subsets (sorted by FACS) using miRCURY RNA Isolation Kit (Exiqon). For micro-RNA, reverse transcription was performed using Universal cDNA Synthesis Kit II (Exiqon), and miR-146a expression was determined by real-time qPCR using ExiLENT SYBR® Green (Exiqon) on ABI7900 apparatus (Applied Biosystems). MiR-146a expression was analyzed in triplicate and normalized to the small-nucleolar RNA 48 (RNU48) housekeeping microRNA. Primers for both miR-146a and RNU48 are obtained from Exiqon.
For mRNA expression, reverse transcription was performed using iScript Reverse Transcription Supermix (Bio Rad). TNF-α expression was determined by real-time qPCR using KAPA SYBR FAST ABI Prism (Kapa Biosystems). Expression of TNF-α was analyzed in triplicate on ABI7900 apparatus (Applied Biosystems) and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping gene. Primer sequences: TNF-α fw: CTG CAC TTT GGA GTG ATC GG; TNF-α rv: GGG TTT GCT ACA ACA TGG GC; HPRT fw: CTT TGC TTT CCT TGG TCA GG; HPRT rv: CAA GGG CAT ATC CTA CAA CAA AC.
For mRNA expression, reverse transcription was performed using iScript Reverse Transcription Supermix (Bio Rad). TNF-α expression was determined by real-time qPCR using KAPA SYBR FAST ABI Prism (Kapa Biosystems). Expression of TNF-α was analyzed in triplicate on ABI7900 apparatus (Applied Biosystems) and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping gene. Primer sequences: TNF-α fw: CTG CAC TTT GGA GTG ATC GG; TNF-α rv: GGG TTT GCT ACA ACA TGG GC; HPRT fw: CTT TGC TTT CCT TGG TCA GG; HPRT rv: CAA GGG CAT ATC CTA CAA CAA AC.
3
RNA Isolation, cDNA Synthesis, and Gene Expression Analysis
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Samples for RNA isolation (n = 9‐11/group) were stored in RNA Stabilization Reagent (RNAlater, Qiagen, Hilden, Germany). Liver RNA was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) with DNase treatment (Sigma‐Aldrich, St. Louis, MO, USA). RNA was converted to cDNA with High Capacity RNA‐to‐cDNA Kit (Applied Biosystems, Foster City, CA, USA). mRNA expression was analyzed with SYBR Green (KAPA SYBR® FAST ABI Prism®; Kapa Biosystems, Woburn, MA) technique using 7300 Real‐Time PCR System (Applied Biosystems). The expression of target genes was quantified relative to geometrical mean of reference genes (β‐actin and ribosomal protein S29). Primers used for quantification are shown in Table 1 .
4
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from the brain tissue of mice using an RNeasy mini kit (Qiagen, Germantown, MD, USA) and was reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher, Waltham, MA, USA) for real-time PCR. cDNA samples were amplified using specific primers (Table S5 ) and KAPA SYBR FAST ABI Prism (KAPA Biosystems, Woburn, MA, USA) using the StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). The target threshold cycle (Ct) was subtracted from the Ct for GAPDH to calculate ΔCt, and relative quantification analysis was performed using the 2−ΔΔCT method25 (link).
5
RNA Extraction and RT-qPCR Analysis of Brain Tissue
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The total RNA in the brain tissue was extracted by using an RNeasy mini kit (Qiagen, Germantown, MD, USA) [28 (link)] and converted into cDNA using a RevertAid First Strand cDNA Synthesis kit (ThermoFisher, Waltham, MA, USA). The cDNA samples in each group were diluted 20-fold with DNase-free water and were subjected to two independent repetitions of real-time PCR with specific primers (Table 1 ) and KAPA SYBR FAST ABI Prism (KAPA Biosystems, Woburn, MA, USA) using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycling conditions of qRT-PCR were 95 °C for 3 min; up to 40 cycles of 95 °C for 3 s, 60 °C for 30 s; and the melt curve stage was 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. The target threshold cycle (Ct) was subtracted from the Ct for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to calculate ΔCt, and a relative quantification analysis was performed via the 2−ΔΔCT method [29 (link)].
6
KAPA SYBR Fast qPCR Amplification Protocol
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KAPA SYBR® Fast Abi Prism® (KAPA Biosystems) was used, to which 10 μL 2X-KAPA SYBR FAST qPCR Master Mix, 0.4 μL of 10 μM primer (Forward), 0.4 μL of 10 μM primer (Reverse) and 1 μL of cDNA Template (×10 dilution) were introduced. Finally, the volume was made to equal 20 μL using either RNase-free water or sterilized deionized water. The mixture was placed into StepOnePlusTM Real-Time PCR System (Applied Biosystems, Thermo Fisher). The condition was set as: preheating at 95 °C for 10 min to activate the enzymes, then entering the cycle of 95 °C, 15 s denaturation, 60 °C 60 s. A total 40 cycles were carried out. On finishing, the 2−ΔΔCT value for each sample was calculated. The primer pairs for RT-PCR used in the study are presented in Supplementary Table S1 .
7
Quantification of CYP19A1 mRNA Levels
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Total RNA was isolated from H295R cells using the AllPrep DNA/RNA Mini Kit (Qiagen) and reverse transcription was accomplished with Superscript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). PCR was performed using 5 ng of cDNA, self-designed (Primer 3.0 web) exon-exon spanning primers (see Table II ) and KAPA SYBR Fast ABI Prism (Kapa Biosystems) mastermix, in a 7500 RealTime PCR System (Thermo Fisher Scientific). Threshold cycle (Ct) values for CYP19A1 were determined in triplicates and presented as average normalized to the average Ct values of the mRNA coding for P0 large ribosomal protein RPLP0 (36B4). Relative fold changes compared to controls were calculated using Pfaffl method.
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