Computational protocol: Textile Hemp vs. Salinity: Insights from a Targeted Gene Expression Analysis

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Protocol publication

[…] Total RNA was extracted and checked as previously described []. The RNA integrity numbers (RINs) were >7.5 for all the samples studied. One microgram of total RNA was retrotranscribed into complementary DNA (cDNA) using the ProtoScript II RTase (NEB, Leiden, The Netherlands) and random hexamers, as previously described [,].The expression was calculated using 3 reference genes (eTIF4E, TIP41 and RAN which geNORM (implemented in qBasePLUS, Biogazelle, Ghent, Belgium) identified as sufficient for data normalization and chosen for the previously reported candidates []). Statistical analyses were carried out using IBM SPSS Statistics v19 (IBM SPSS, Chicago, IL, USA) (t-test for independent samples). [...] Primers were designed using Primer3Plus [] and verified with the OligoAnalyzer 3.1 tool from Integrated DNA Technologies (Leuven, Belgium) (http://eu.idtdna.com/calc/analyzer). Primer efficiencies were calculated via RT-qPCR using a serial five-fold dilution of cDNA (25, 5, 1, 0.2, 0.04, 0.008 ng/µL). The primer sequences, amplification efficiencies and R2 have either been previously published (eTIF4E, TIP41, F-BOX, RAN, PAL, CAD, 4CL, FLA1-3-6-8-10, EXPA8, CesA4-7-8 [,]), or are indicated in . The identification numbers (IDs) of the genes actin depolymerizing factor 5 ADF5, ethylene response factor ERF1, auxin-responsive protein IAA11, calcium-dependent lipid-binding family protein isoform 2 CALB2, calcium-dependent lipid-binding family protein isoforms 4 CALB4-1,-2,-3, heat shock protein 70 HSP70-1 and -2, heat shock protein HSP81.4, and gibberellin receptor 1b GIBB-REC, histidine kinase 4-like HK4 have been previously described []. […]

Pipeline specifications

Software tools SPSS, Primer3, OligoAnalyzer
Applications Miscellaneous, qPCR
Organisms Cannabis sativa
Chemicals Calcium, Sodium Chloride