Total RNA of PBMCs from patients and HDs was isolation using TRIzol reagent (TaKaRa, China) according to the manufacturer’s instructions. All RNA preparation steps were performed under RNase-free coding. RNA concentration was determined using a NanoDrop 2000 spectrophotometer and RNA quality was verified by 1.5% non-denaturing agarose gel electrophoresis. Reverse transcription of RNA was carried out with the PrimeScript RT Master Mix system (TaKaRa).
Primescript rt master mix system
Manufactured by Takara Bio
Sourced in Japan, China
The PrimeScript RT Master Mix system is a reagent kit designed for reverse transcription of RNA to cDNA. The kit contains a proprietary reverse transcriptase enzyme and necessary buffers and reagents to perform the reverse transcription reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green qpcr assay, by Takara Bio (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy spin column, by Qiagen (1 mentions)
Sybr master mix, by Roche (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Sybr premix, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
10 protocols using primescript rt master mix system
1
Total RNA Isolation and Reverse Transcription
2
RNA Extraction and qPCR Analysis
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RNA from tissues and cells was extracted by TRI-reagent (Sigma, USA) according to the manufacturer’s protocol. Then the RNA was measured with a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was then synthesized with PrimeScript RT Master Mix System (Takara, China) following the manufacturer’s protocol. Real-time qPCR of the reverse transcription products was determined with SYBR Green qPCR assay (Takara, China), analyzed through the 7500 Real-time PCR System (ABI, USA) and normalized with GAPDH or U6, respectively. The relative expression levels of RNAs were calculated using the comparative 2-ΔΔCT method. All experiments were performed at least three times. The primers used for RT-qPCR are listed in Table 2 .
3
CYP1B1 Expression in Prostate Tissue
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This analysis included 218 patients whose frozen adjacent normal prostate tissue was available for RNA extraction. Tissue specimens were collected during surgery and stored at −80 °C. The TRIzol reagent (Invitrogen, CA) was used to isolate total RNA. cDNA was synthesized using PrimeScriptRT Master Mix system (TAKARA, Osaka, Japan). Quantitative PCR was carried out using ABI 7900 Real-Time PCR System (Applied Biosystems, CA). The sequences of the CYP1B1 specific primers were forward, GCTGCAGTGGCTGCTCCT, and reverse, CCCACGACCTGATCCA AT TCT. The negative controls for each primer consisted of a reaction with no cDNA added. Quantification was achieved by the use of a standard curve for CYP1B1 and normalization of the corresponding transcripts by the ΔΔCt method and the use of the housekeeping gene GAPDH.
4
mRNA Expression Analysis of NSCLC
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Non–small cell lung cancer tumor tissues were dissected and evaluated by two pathologists after surgical removal, and the leftover tumors tissues were transferred into liquid nitrogen immediately after resection and stored at −80°C until use at the Department of Pathology and Tissue Bank. For the present study, 170 tumor tissue samples were randomly selected for mRNA expression analysis. Both RNA and DNA were extracted from tumor tissue samples with the TRIzol reagent (Invitrogen, Carlsbad, CA) and the DNA kit (Tiangen, Beijing, China). RNA samples were used to synthesize complementary DNA with the PrimeScript RT Master Mix system (Takara, Otsu, Japan), as previously described [23] (link). The DNA samples were used for genotyping as mentioned above.
5
Extracting and Quantifying RNA from PBMCs
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Total RNA was extracted from the separated PBMCs from 70 mCRPC patients and 20 healthy male volunteers using the TRIzol Reagent (Invitrogen, USA). RNA samples extracted from LNCaP cells served as a positive control. All RNA preparation and handling procedures were performed under RNase-free conditions in a laminar flow hood. Optical density measurements at 260 and 280 nm were used to quantify and assess the purity of the RNA. Reverse transcription of RNA was carried out with the PrimeScript RT Master Mix system (TaKaRa, Dalian, China). Complementary DNA was synthesized from 2 μg of total RNA isolated from PBMCs in a total volume of 20 μl, according to the manufacturer's instructions.
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from the cultured cells using RNeasy spin columns (Qiagen, Valencia, CA, USA). After reverse transcription into complementary DNA using PrimeScript RT Master Mix system (TaKaRa Bio, Shiga, Japan), real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Gene transcripts were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression and quantified as relative expression levels.
7
RNA Extraction and RT-qPCR Analysis
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TRIzol reagent (Thermo Fisher Scientific, USA) was used to extract total RNA of cells. A PrimeScript RT Master Mix system (Takara, Japan) was used to synthesize cDNA. Real-time PCR was performed using SYBR Master Mix (Roche, Switzerland). Primers for real-time PCR are listed in Additional file 1 : Table S1.
8
Quantitative gene expression analysis
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Total RNA was extracted from primary floral bud clusters of the main inflorescence shoot (up to 10 per biological replicate), collected, and ground in liquid nitrogen. RNA extraction and clean-up was performed using an RNeasy kit (Qiagen) with an in-column DNase I digest. PrimeScript RT Master Mix system (Takara) was used for first-strand cDNA synthesis. The subsequent qPCR reaction mix was prepared using a FastStart Essential DNA Green Master (Roche). qPCR was performed with a Light Cycler 480 (Roche) instrument using the Light Cycler 480 release 1.5.1.62 SP software (Roche). Relative transcript abundance across 3 biological replicates was calculated using the comparative CT method; statistical analyses were performed using a 2-tailed Student's t test. The sequences of primer pairs used for genes of interest and the reference gene TUBULIN2 are listed in Supplemental Table S2.
9
CD44 Gene Expression Analysis by qRT-PCR
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TRIzol reagent (Invitrogen, CA, USA) was used to extract total RNA from cells. The Prime ScriptTM RT Master Mix system (TaKaRa) was used to reverse mRNA into complementary DNA (cDNA). The relative expression level of CD44 was determined by real-time PCR analysis using SYBR Premix (Applied Biosystems, CA, USA) on an ABI 7500 Real-Time PCR system (Applied Biosystems). GAPDH was used for normalization. The amplification reaction was set as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Primer sequences were as follows: GAPDH, forward primer 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer 5′-GAAGATGGTG-ATGGGATTTC-3′; CD44, forward primer 5′-ACCCCAACTCCATCTGTGC-3′, reverse primer 5′-TTCTGGACATAGCGGGTG-3′.
10
RNA Extraction and cDNA Synthesis
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Total RNA was isolated from 60 samples by using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the steps in the manufacturer's protocol. Two percent (w/v) agarose gel electrophoresis was performed to validate the RNA quality, and a NanoDrop 2000 c spectrophotometer (Thermo Scienti c, Wilmington, USA) was used to determine the RNA concentration. Then 1 µg of total RNA was used to synthesize cDNA by applying the PrimeScript TM RT Master Mix system (TaKaRa, Dalian, China), following the steps described in the manufacturer's instructions.
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