Computational protocol: Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)

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Protocol publication

[…] To search for HKG sequences from L. decemlineata transcriptome data, a reciprocal BLAST hits approach was used. The HKGs from other insect species in GenBank were downloaded from NCBI ( http://www.ncbi.nlm.nih.gov/), queried individually to L. decemlineata transcriptome using the TBLASTN program with a permissive E-value cutoff of 10-3 to get the hits. And then, each of the queried hits was compared back against non-redundant database of NCBI by the BLASTX program (E-value <10-3) to determine whether the original sequence was one of the hits. The selected HKG sequences were listed in Table  .The unigenes of selected nine HKGs were assembled and clustered from short reads, there were inevitably issues with clone contamination and mix-up. Reverse transcriptase PCR (RT-PCR) was performed to authenticate the HKGs using the primers listed in Table  . The components of PCR reaction buffer were 2.5 mM of dNTP, 10 mM of each primer, 25 mM of MgCl2, 5 U/μL of Ex-Taq DNA polymerase (Takara Bio, Dalian, China), in a total volume of 25 μL. Thermal cycling conditions of RT-PCR were available from the authors upon request. The amplified products were separated by electrophoresis on 1.5% agarose gel and purified using the Wizard® PCR Preps DNA Purification System (Promega). Purified DNA was ligated into the pGEM®-T easy vector (Promega) and several independent subclones were sequenced from both directions. The nucleotide sequences obtained after the sequence analysis were submitted to GenBank database (Accession No. KC190026- KC190034). [...] The qRT-PCR primers were designed using Beacon Designer 7 (Premier Biosoft International, Palo Alto, Calif., USA), and were given in Table  . The qRT-PCR reactions were performed using SYBR Premix Ex Taq (Perfect Real Time) (Takara Co., Otsu, Japan) and ABI Real-Time 7300 PCR system (Applied Biosystems) according to the manufacturer’s protocol. The reaction mixture consisted of 2 μL of cDNA template (corresponding to 0.9 ng of the starting amount of RNA), 10 μL of SYBR Premix Ex Taq (Takara), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 0.4 μL of Rox Reference Dye (50×) in a final reaction volume of 20 μL. A reverse transcription negative control (without reverse transcriptase) and a non-template negative control were included for each primer set to confirm the absence of genomic DNA and to check for primer-dimer or contamination in the reactions, respectively. The qRT-PCR protocol included an initial step of 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and then annealed at 60°C for 31 sec, followed by one cycle of 95°C for 15 sec, 60°C for 60 sec, and 95°C for 15 sec. PCR amplicons were subjected to melting curve analysis. The specificity of the qRT-PCR reactions was monitored with melting curve, analyzing by SDS software (version 1.4) and gel electrophoresis. Amplification efficiencies were determined by a 10-fold dilution series of template. All experiments were repeated in triplicate. [...] The raw Ct values were obtained using the SDS software of ABI 7300 (version 1.4). The algorithms including geNorm [], BestKeeper [] and NormFinder [] were used to analyze the stability of selected HKGs, strictly following the manuals of the algorithms. […]

Pipeline specifications

Software tools Beacon Designer, BestKeeper, NormFinder
Application qPCR
Organisms Solanum tuberosum
Chemicals Adenosine Diphosphate