Total RNA was isolated from tumor tissue using Purelink RNA Minikits (Ambion, Life Technologies) according to the manufacturer’s instructions. The quality and quantity of RNA samples were measured using a spectrophotometer (Amersham Biosciences, Little Chalfont, UK). An A260/A280 ratio of 1.8–2.0 for RNA was considered suitable for real time qPCR. cDNA was prepared using the Reverse Transcriptase Mix (Qiagen Ltd.). Quantitative PCR was carried out in accordance with the RT2 Profiler PCR array instructions. Cancer Inflammation and Immunity Crosstalk PCR Arrays (Qiagen) were used to analyze the expression of 84 immune-related genes, a subset of which (CTLA-4, FOXP3, granzyme B, and IDO1) were selected for further study by real time quantitative PCR using the Taqman probes for specific genes (Applied Biosystems) according to manufacturer’s instructions. The relative mRNA values were calculated by double delta method and normalized to the level of housekeeping gene GusB.
Reverse transcriptase mix
Manufactured by Qiagen
Reverse Transcriptase Mix is a laboratory reagent designed for the conversion of RNA into complementary DNA (cDNA) during the reverse transcription process. The core function of this product is to facilitate the enzymatic conversion of RNA into single-stranded cDNA, which can then be used for various downstream applications, such as gene expression analysis, RT-PCR, and cDNA library construction.
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Lab products found in correlation
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Taqman probes for specific genes, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Evagreen, by Biotium (1 mentions)
Hispec buffer, by Qiagen (1 mentions)
Nucleics mix, by Qiagen (1 mentions)
2 protocols using reverse transcriptase mix
1
Immune Gene Expression Analysis in Tumors
2
miR-192a Expression Analysis by qRT-PCR
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According to the manufacturer's instructions (miScript PCR Starter Kit, cat. No. 218193; Qiagen), RNA was reverse transcribed in a final volume of 20 ml containing 4 μl 5 × miScript HiSpec Buffer, 2 μl 10 × miScript Nucleics Mix, 2 μl miScript Reverse Transcriptase Mix, 7 μl RNase-free water, and 5 μl template RNA. The mix was incubated at 37°C for 60 min and 95°C for 5 min, and then held at 4°C. Real-time quantification was then performed using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA).
Each real-time PCR included 0.5 ml universal reverse primer, 0.5 ml sense primer, 1 ml EvaGreen (Biotium, Hayward, CA, USA), 1 U ml−1 TaqMan (Biotium), and 1 ml reverse transcription product. The reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. U6 snRNA was used as the endogenous control gene to normalize the miR-192a content among different samples. The expression of miR-192a relative to U6 snRNA was determined using the 2−ΔCt method. The tests were repeated three times per experiment.
Each real-time PCR included 0.5 ml universal reverse primer, 0.5 ml sense primer, 1 ml EvaGreen (Biotium, Hayward, CA, USA), 1 U ml−1 TaqMan (Biotium), and 1 ml reverse transcription product. The reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. U6 snRNA was used as the endogenous control gene to normalize the miR-192a content among different samples. The expression of miR-192a relative to U6 snRNA was determined using the 2−ΔCt method. The tests were repeated three times per experiment.
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