Nucleotrap mrna kit

Poly(A) fractionation was performed as previously described [44 (link)]. In brief, NucleoTrap mRNA kit (Clontech) was used to fractionate Poly(A) plus and Poly(A) minus fractions following by extraction, RT and qPCR.

Fresh samples of C. faberi, including young leaves, root, and flowers in different stages (flower bud stage, blooming stage and withered stage) were collected and ground into powder after freezing with liquid nitrogen. The total RNA was extracted according to the Trizol Extraction Protocol (Takara, Dalian, China). The extracted total RNA (~ 200 μg) was diluted in 100 μL DEPC ddH₂O, incubated at 65 °C for 2 min and stored on ice. Total RNAs from different tissues and different stages were mixed together for the subsequent purification. The mRNA was purified via the poly A tail using the NucleoTrap mRNA kit (Clontech, CA, USA) according to the manufacturer’s instructions. After washing with 200 μL of washing buffer twice, 10~20 μL 10 mM Tris-HCl (pH 7.5) was added to elute the mRNA at 80 °C for 2 min. The first-strand cDNA was synthesized using the SMART III reverse-transcriptase kit (Takara, Dalian, China). The reverse-transcriptase reaction was terminated at 75 °C for 10 min, after which 1 μL RNase H (2 Units) was added and incubated at 37 °C for 20 min to digest the redundant mRNA.

RLM-RACE was performed using the GeneRacer Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions with minor modifications. Total RNA from developing fibers at 0, 3, 8 and 16 DPA was combined for mRNA isolation. Poly(A) mRNA was purified from total RNA using NucleoTrap mRNA Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The GeneRacer RNA Oligo adapter was directly ligated to mRNA without calf intestinal phosphatase and tobacco acid pyrophosphatase treatment, which would have restricted analysis to full length mRNA. The GeneRacer Oligo dT primer was then used to synthesize first-strand cDNA. Two sets of reactions were performed: 1) with the GeneRacer 5’ Primer and gene-specific primers; and 2) with the GeneRacer 5’ Nested Primer and gene-specific nested primers (Additional file 7). After amplification, 5’RACE products were gel-purified and cloned into the pCR 4-TOPO vector, and approximately 10 independent clones were randomly chosen and sequenced.