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Sybr green qpcr assay

Manufactured by Takara Bio
Sourced in China

The SYBR green qPCR assay is a quantitative real-time PCR reagent that utilizes the SYBR green dye for the detection and quantification of DNA sequences. The SYBR green dye binds to double-stranded DNA, and the fluorescence emitted during the PCR amplification process is used to monitor the accumulation of the target DNA sequence.

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40 protocols using sybr green qpcr assay

1

RNA Extraction and miRNA-mRNA Analysis

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Total RNA was extracted using Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the provided instructions. The miR-516a-3p level was investigated by TaqMan miRNA assays (Thermo Fisher Scientific) according to the manufacturer’s protocol. miRNA U6 was used for normalization. ABCC5 mRNA level was studied by SYBR green qPCR assay (Takara, Dalian, China). β-Actin was used as control.
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2

Quantification of miRNA and mRNA Expression in Colorectal Cancer

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Total RNA was extracted from CRC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression levels of miRNAs were detected using the Hairpin-it miRNAs qPCR Kit (Genepharma, Shanghai, China). The expression of RNU6B served as an endogenous control. iASPP expression was measured using a SYBR Green qPCR assay (Takara, Dalian, China). The data were processed according to the 2−ΔΔCT method; the relative expression of miR-124 was calculated with the formula, 2−(CTmiRNA−CTRNU6B). The primers are shown in Supplementary Table 1.
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3

RNA Extraction and miRNA/mRNA Expression Analysis

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Total RNA was extracted from CRC cells using TRIzol reagent (Invitrogen, CA, USA). Total RNA was extracted from tissue samples following the protocol by Helen Pearson [23 (link)]. The expression levels of miR-124 and miR-155 were detected using the Hairpin-it™ miRNAs qPCR Kit (Genepharma, Shanghai, China). The expression of RNU6B served as an endogenous control. IASPP, p63 and STAT1 expression was measured using a SYBR Green qPCR assay (Takara, Dalian, China). The data were processed according to the 2−ΔΔCT method. The primers were shown in Supplementary Table 1.
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4

Quantitative Analysis of miR-101 and KDM1A Expression

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Total RNA was extracted from tissue and cell specimens using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. The detection of qRT-PCR was performed by using SYBR green qPCR assay (Takara, People’s Republic of China). The primers of miR-101 and U6 were purchased from Riobio Biotech Corporation (Guangzhou, People’s Republic of China). The primers of KDM1A were 5′-CTGATGCAGGCCATCAAGT-3′ (forward) and 5′-TCTCCAGGAAATGCATTGGT-3′ (reverse). The primers of GAPDH were 5′-TGGTGGACCTCATGGCCTAC-3′ (forward) and 5′-CAGCAACTGAGGGCCTCTCT-3′ (reverse). PCR reaction condition was as follow: 95°C for 15 minutes, followed by 40 cycles of 94°C for 15 seconds, 60°C for 30 seconds and 70°C for 30 seconds. Relative expression level was compared by the 2−∆∆CT method.
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5

Quantifying mRNA Expression Using qPCR

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Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen). The expression of mRNA was measured using a SYBR Green qPCR assay (Takara). The expression of GAPDH served as an endogenous control. The 2−ΔCT method was applied for data processing.
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6

Measuring miRNA and mRNA Levels via SYBR Green qPCR

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Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen). The miRNA and mRNA levels were measured using a SYBR Green qPCR assay (TaKaRa, Dalian, China) following the methods described before34 (link). The expression of GAPDH (reference for mRNA determination) or RNU6B (reference for miRNA determination) served as an endogenous control. The sequences of the PCR primers are presented in Supplementary Table S1. The 2−ΔCT method was applied for data processing.
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7

Quantification of miRNA-101 and MALAT1 Expression

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Total RNA was extracted from cells using Trizol reagent (Invitrogen, CA, USA) and detected mature miR-101 expressions in cells using a Hairpin-it TM miRNAs qPCR kit (Genepharma, Shanghai, China). Primers for miR-101 were forward, 5’-GGCGTACAGTACTGTGATA-3’. Expression of RNU6B was used as an endogenous control. Primers for RNU6B were forward, 5’-CTCGCTTCGGCAGCACA-3’ and reverse, 5’-AACGCTTCACGAATTTGCGT-3’. MALAT1 expression was measured by SYBR green qPCR assay (Takara, Dalian, China). Primers for MALAT1 were forward, 5’- AAGAAGCCGAAATAAATGAG-3’ and reverse, 5’-AGCCCACAGGAACAAGTC- 3’. Data were processed using 2-ΔΔCT method.
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8

Quantifying MIAT and miR-34a Expressions

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Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, United States). Total RNA was reverse transcribed using a transcription kit (Invitrogen) to get total cDNA as templates for MIAT detection, or transcribed with TakaraTM microRNA transcription kit to get cDNA as templates for miR-34a detection. The expression of MIAT was measured by SYBR green qPCR assay (Takara, Dalian, China) according to manufacturers’ instructions. Expression of β-actin was used as an endogenous control. MiR-34a expression was detected using a Hairpin-it TM miRNAs qPCR kit (Genepharma, Shanghai, China) according to manufacturers’ instructions. Expression of RNU6B was used as an endogenous control. QPCR was performed at the condition: 95.0°C for 3 min, and 39 circles of 95.0°C for 10 s and 60°C for 30 s. Data were processed using 2-ΔΔCT method. The primers were used as following: 5′- TCCCATTCCCGGAAGCTAGA -3′(forward), 5′- GAGGCATGAAATCACCCCCA -3′(reverse) for MIAT. 5′- TTGTTACAGGAAGTCCCTTGCC-3′(forward), 5′- ATGCTATCACCTCCCCTGTGTG-3′(reverse) for β-actin. Sequence of miRNA and references using in current study are: miR-34a: UGGCAGUGUCUUAGCUGGUUGU; RNU6B: CGCAAGGAUGACACGCAAAUUCGUGAAGCGUUCCAUAUUUUU. The reverse primers were also used in the Hairpin-it TM miRNAs qPCR kit.
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9

Quantifying BANCR Expression in Cells

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Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, CA, USA). BANCR expressions were measured by SYBR green qPCR assay (Takara, Dalian, China) in triplicate. Primer sequences were as follows: BANCR, forward: ACAGGACTCCATGGCAAACG, reverse: ATGAAGAAAGCCTGGTGCAGT; GAPDH, forward: ACCACAGTCCATGCCATCAC, reverse: TCCACCACCCTGTTGCTGTA. Data were processed using 2-ΔΔCT method and normalized to GAPDH expression.
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10

Gene Expression Analysis of Clinical Samples

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Total RNA from clinical tissues and stably transfected HTR-8/Svneo and BeWo cells was isolated using Trizol (Invitrogen, USA). ImProm-II Reverse Transcription System (Promega, USA) was then used to generate first-strand cDNA. SYBR Green qPCR assay (Takara, Dalian, China) and gene-specific primers were used for qRT-PCR with GAPDH or U6 used for normalization following the manufacturer’s protocol. The relative expression levels of genes were calculated according to the 2−ΔΔCt method [16 (link)]. Each sample was tested in triplicates for statistical analysis.
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