Total RNA from MKN45 and MKN45-R was isolated using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. TRIzol (Invitrogen, USA) was used to extract total RNA from gastric cancer tissues and adjacent normal tissues. SYBR Green PCR Kit (Takara, Japan) and ABI 7500 System were used to detect the expression of SNHG6 and miR-325-3p. miDETECT A Track Kit (RiboBio, China) was used to detect the expression of miR-325-3p. As for miR-325-3p, U6 was employed for the normalization control. As for SNHG6, GAPDH was used for normalization control. SYBR Premix Ex Taq (Takara, Japan) was used for the qRT-PCR.
Midetect a track kit
Manufactured by RiboBio
Sourced in China
The MiDETECT A Track Kit is a laboratory instrument designed for the detection and quantification of miRNA expression. The kit utilizes real-time PCR technology to provide accurate and reliable results. Its core function is to analyze small RNA samples and generate quantitative data on miRNA levels.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr kit, by Takara Bio (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7500 fast dx real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand dna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Toyobo (1 mentions)
Genomic dna purification kit, by Promega (1 mentions)
6 protocols using midetect a track kit
1
SNHG6 and miR-325-3p Expression Analysis
2
Comprehensive RNA Extraction and Analysis
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Total RNA extraction from tissue and cell samples was carried out with TRIzol (Invitrogen, USA) following the kit’s protocol. LncRNAs and mRNAs were assessed with a SYBR Green PCR Kit (Takara, Japan), using GAPDH as an internal control gene. A miDETECT A Track Kit (RiboBio, China) was utilized to detect microRNAs, with U6 for normalization. The 2−△△Ct method was utilized for data analysis. Primer sequences were listed in Supplementary Table 2 .
3
Quantitative Analysis of lncRNAs and miRNAs
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Total RNA was extracted from tissue and cell specimens with TRIzol (Invitrogen, USA) as directed by the manufacturer. A SYBR Green PCR Kit (Takara, Japan) was utilized to detect lncRNAs and mRNAs. GAPDH was utilized to normalize transcript levels. MicroRNAs were assessed with a miDETECT A Track Kit (RiboBio, China), using U6 as a reference. The relative quantification of RNAs was performed by the 2−△△Ct method. The sequences of the primers used in the study were listed in Table 1 .
QRT-PCR primer
sequences
| Gene | Forward | Reverse |
|---|---|---|
| GACAT3 | 5′-CTTCCGGAGCAGGTCTGAGT-3′ | 5′-CTTTCCCTGCAGAGACCAGT-3′ |
| FoxM1 | 5′-TATTCACAGCATCATCACAGC-3′ | 5′-GAAGGCTCCTCAACCTTAACCT-3′ |
| miR-149-5p | 5′-GGCTCTGGCTCCGTGTCTT-3′ | 5′-CAGTGCAGGGTCCGAGGTATT-3′ |
4
Comprehensive Analysis of RNA Expression
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Total RNA was extracted from cells and tissues using the TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). The subcellular fractions of NSCLC cells were separated using the
PARIS Kit (Ambion, Austin, TX, USA). RevertAid™ First Strand DNA Synthesis Kit (Thermo
Fisher Scientific, Waltham, MA, USA) was used for reverse transcription, and quantitative
real-time polymerase chain reaction (qRT-PCR) was executed using an SYBR Green PCR Kit
(Toyobo, Osaka, Japan) on the 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems,
Foster City, CA, USA). β-actin was considered as a control for
normalization. MicroRNA detection was conducted using a miDETECT A Track Kit (RiboBio,
Guangzhou, China). The small nuclear RNA U6 expression was employed as a
control for normalization. The primers for this research were designed using Primer
Premier 5 software. The sequences are presented inTable S1 (See Supplementary Online
Information atwww.celljournal.org ).
Carlsbad, CA, USA). The subcellular fractions of NSCLC cells were separated using the
PARIS Kit (Ambion, Austin, TX, USA). RevertAid™ First Strand DNA Synthesis Kit (Thermo
Fisher Scientific, Waltham, MA, USA) was used for reverse transcription, and quantitative
real-time polymerase chain reaction (qRT-PCR) was executed using an SYBR Green PCR Kit
(Toyobo, Osaka, Japan) on the 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems,
Foster City, CA, USA). β-actin was considered as a control for
normalization. MicroRNA detection was conducted using a miDETECT A Track Kit (RiboBio,
Guangzhou, China). The small nuclear RNA U6 expression was employed as a
control for normalization. The primers for this research were designed using Primer
Premier 5 software. The sequences are presented in
Information at
5
Quantifying lncRNA, mRNA, and miRNA Levels
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Total RNA was isolated from the tissue samples and cells by TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. lncRNA and mRNA detection was performed using a SYBR Green PCR Kit (Takara, Japan) with an ABI 7500 System. GAPDH expression was employed as a control for normalization. MicroRNA detection was performed using a miDETECT A Track Kit (RiboBio, China). The expression of the small nuclear RNA U6 was used as a control for normalization. For SNHG6 copy number detection, genomic DNA was isolated using a Genomic DNA Purification Kit (Promega, USA). QRT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan) with an ABI 7300 System. POLR2A, RPP14, andTBX15 expression levels were employed for normalization. These three loci are housekeeping genes, and their copy numbers are stable across the population. A sample with a mean expression of SNHG6 relative to these reference genes greater than 1.5 is defined as copy number gain. A sample with a mean expression of SNHG6 relative to these reference genes less than 0.6 is defined as copy number loss. Each experiment was repeated at least three times. Primers are listed in Additional file 1 : Table S1.
6
Comprehensive RNA Expression Analysis
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Total RNA was extracted from tissue and cell specimens with TRIzol (Invitrogen, USA) as directed by the manufacturer. A SYBR Green PCR Kit (Takara, Japan) was utilized to detect lncRNAs and mRNAs. GAPDH was utilized to normalize transcript levels. MicroRNAs were assessed with a miDETECT A Track Kit (RiboBio, China), using U6 as a reference. The relative quanti cation of RNAs was performed by the 2 -△△Ct method. The sequences of the primers used in the study were as follows: GACAT3 5′-CTTCCGGAGCAGGTCTGAGT-3′ (forward), and 5′-CTTTCCCTGCAGAGACCAGT-3′ (reverse); miR-149-5p, 5′-GGCTCTGGCTCCGTGTCTT-3′(forward), and 5′-CAGTGCAGGGTCCGAGGTATT-3′(reverse); FoxM1, 5′-TATTCACAGCATCATCACAGC-3′(forward) and 5′-GAAGGCTCCTCAACCTTAACCT-3′ (reverse).
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