Crna amplification and labeling kit

Total RNA samples at different fiber developmental stages from 13–25 DPA in TM-1 and the im mutant were extracted using the CTAB-sour phenol extraction method [69 ]. The isolated RNA was purified using the NucleoSpin® RNA clean-up kit (MACHEREY-NAGEL, Düren, Germany) and checked for quantity and integrity by spectrophotometry and 1.2% agarose formaldehyde denaturing gel electrophoresis. RNA amplification and labeling were performed using the cRNA Amplification and Labeling Kit (CapitalBio, Beijing, China) per the manufacturer’s protocol. Briefly, total RNA was reversely transcribed into double-stranded cDNA using the CbcScript enzyme. Subsequently, cRNA was synthesized from cDNA products using the T7 Enzyme Mix in the cRNA Amplification and Labeling Kit and purified using the NucleoSpin® RNA clean-up kit. After the cRNA was synthesized, the new cDNA was generated with random primers from the cRNA using CbcScript II enzyme in the kit. Finally, the transcription products were purified with the Nucleospin® Extract II Kit (MACHEREY-NAGEL). The cDNA sample from TM-1 and im mutant at the same fiber developmental stage was labeled with Cy5 and Cy3-dCTP, respectively. And Cy5 and Cy3-dCTP interchanged to be used to label cDNA samples in the dye swap experiment.

The cRNA amplification and labeling Kit (CapitalBio, China) was used to amplify and label RNA following the manufacturer’s protocol. cDNA samples from XinWX and XinFLM collected during the same fiber developmental stage were labeled with Cy3 and Cy5-dCTP, respectively, and these dyes were interchanged to label cDNA samples in a dye swap experiment.

Complementary DNA (cDNA) labeled with Cy3-dCTP (for primary HCC) or Cy5-dCTP (for recurrent HCC) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction using a cRNA Amplification and Labeling Kit (CapitalBio, Beijing, China) [22 (link)]. The labeled cDNA was hybridized with CapitalBio Technology Human LncRNA Array V4 containing probes inspecting about 41,000 human lncRNAs and approximately 34,000 human mRNAs. Microarray data were analyzed using the GeneSpring software V13.0 (Agilent, Santa Clara, CA, USA). Genes with an absolute fold change value ≥2 and a Benjamini-Hochberg corrected P- value ≤0.05 were treated as differentially expressed genes. Hierarchical clustering analysis was performed using Cluster 3.0 software (Stanford University, CA, USA).