The mRNA level of each gene was measured via qRT-PCR. Moreover, the change in 3′ UTR lengths of target genes was also examined via qRT-PCR for increasingly distal regions of target gene transcript 3′ UTRs relative to the level of the coding sequence of each of the genes. RNA was isolated using a total RNA isolation kit, including an on-column DNase treatment (Norgen). cDNA synthesis was carried out using the SuperScript III reverse transcriptase kit using a mixture of oligodT and random hexamers for priming (Life Technologies). qRT–PCR was conducted with Fast SYBR Green master mix (Applied Biosystems), and fluorescence was monitored using a 7900HT Fast real-time instrument (Applied Biosystems). Data were analyzed using the ΔΔCt method. Endogenous control transcripts were used for normalization. Statistical significance was determined using a one-tailed Student’s t-test. The sequences of the primers used for all qRT–PCR assays are listed in Additional file 7 : Table S6.
Random hexamers for priming
Manufactured by Thermo Fisher Scientific
Random hexamers are short DNA sequences consisting of six random nucleotides that are used as primers for various molecular biology applications, such as cDNA synthesis and DNA amplification. They provide a versatile and unbiased approach to priming target sequences without the need for specific sequence information.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
7900ht fast real time instrument, by Thermo Fisher Scientific (3 mentions)
Oligo dt, by Thermo Fisher Scientific (1 mentions)
Dnase treatment, by Norgen Biotek (1 mentions)
3 protocols using random hexamers for priming
1
Quantitative Analysis of Gene Expression
2
Gene Expression Quantification via qPCR
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The mRNA levels of each gene were measured via qPCR. Following procedures previously described (50 (link)), RNA was isolated using a total RNA isolation kit, including an on-column deoxyribonuclease treatment (Norgen). cDNA synthesis was carried out using the SuperScript III Reverse Transcriptase Kit using a mixture of oligo(dT) and random hexamers for priming (Life Technologies). qPCR was conducted with Fast SYBR Green Master Mix (Applied Biosystems), and fluorescence was monitored using a 7900HT Fast real-time instrument (Applied Biosystems). Data were analyzed using the ΔΔCt method. The endogenous control transcripts were used for normalization. Statistical significance was determined using a one-tailed Student’s t test. The sequences of the primers used for all qPCR assays are in table S3.
3
Quantitative Real-Time PCR Protocol
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RNA was isolated using a total RNA isolation kit, including an on-column DNase treatment (Norgen). cDNA synthesis was carried out using the SuperScript III reverse transcriptase kit using a mixture of oligodT and random hexamers for priming (Life Technologies). qRT–PCR was carried out using Fast SYBR Green master mix (Applied Biosystems), and fluorescence was monitored using a 7900HT Fast real-time instrument (Applied Biosystems). Data were analyzed using the ΔΔCt method. The endogenous control transcripts that were used for normalization are indicated in each figure. Statistical significance was determined using a one-tailed Student's t-test. The sequences of the primers used for all qRT–PCR assays are in Supplemental Table S2.
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