Trizol plus purification kit

Genomic DNA samples were extracted from mixed stages of C. elegans or L3s of H. contortus using the EasyPure Genomics DNA Kit (TransGen Biotech, China). Total RNA samples were isolated separately from egg, L1, L2, iL3, female and male fourth-stage larvae (L4s), and female and male adult stages of H. contortus using the TRIzol Plus Purification kit (Life Technologies, USA). RNA yields and quality were verified by spectrophotometric (NanoDrop Technologies) and by electrophoretic analysis, respectively. RNA was treated with RQ1-RNase-Free DNase (Promega, USA). Following isolation, nucleic acid samples were immediately frozen and stored at −80 °C.

RNA extraction was performed using the Trizol Plus Purification Kit (Invitrogen) according to the manufacturer’s instructions. On-column DNA digestion (RNase-Free DNase Set, Qiagen) was used to ensure the absence of DNA from the samples. The quantification and quality analysis of RNA was performed on a Bioanalyzer 2100 (Agilent, Santa Clara, California).

Wild-type embryos were dissected in PBS with calcium and magnesium. The embryos were then incubated in pancreatin/trypsin in calcium- and magnesium-free PBS for dissection. The surface ectoderm and neural ectoderm at the forebrain level, which does not include cephalic mesenchymal cells, were isolated using tungsten needles. The numbers of embryos used for three independent biological replicate experiments were as follows: replicate 1, surface ectoderm 1 (SE1) n = 109, neural ectoderm 1 (NE1) n = 46; replicate 2, SE2 n = 117, NE2 n = 43; and replicate 3, SE3 n = 114, NE3 n = 45. Isolated tissues were stored at − 80 °C after being soaked in RNAlater (Applied Biosystems). Collected tissues were purified using a TRIzol-plus purification kit (Invitrogen, cat. no. 12183-555). The quality of purified RNA samples was confirmed using Experion (BioRad). cRNA was prepared using the illumine Total Prep RNA Amplification Kit (Illumina). Samples were hybridized to a MouseWG6-v2 array (Illumina) and scanned with a BeadArray Reader (Illumina). Raw data were analyzed using Bead Studio software (Illumina), and pairwise comparisons were done between the SE and NE populations for each of the three biological replicates. We noted that there was good correspondence between replicated genes with a two-fold change. The accession number for the microarray data reported in this paper is GSE67977.

The manufacturer’s instructions for the Fugene 6 transfection reagent were followed in order to transfect the plasmid into the brain glioma, SP2/0, L929 and HeLa cells (all obtained from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences, Shanghai, China). The primers were designed according to the sequence of the recombinant pINV-HPV16E6/7 plasmid. The sequences of the upstream forward and the downstream reverse primers were 5′-CACAGGAGCGACCCAGAAAGTTA-3′ and 5′-GCTGGG TTTCTCTACGTGTTCTT-3′, respectively. It was estimated that the amplified DNA was 438 bp in length. The mRNA that was transfected into the brain glioma, SP2/0, L929 and HeLa cells was extracted according to the manufacturer’s instructions for the TRIzol Plus Purification kit (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was amplified using the Super Script kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. A total of 10 μl cDNA was mixed with 4 μl 10X PCR buffer, 2 μl (25 mM) MgCl₂, 0.5 μl (100 pmol) of each primer, 32.7 μl distilled water and 0.3 μl (2.5 U) Taq DNA polymerase. The PCR reaction conditions were as follows: 35 cycles of 30 sec at 94°C, 30 sec at 50°C and 2 min at 72°C. The amplified products were sequenced to confirm pINV-HPV16E6/7 DNA (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.).

RNA isolation from samples was performed at three ASC culture stages: day 1 (early control), day 30 of culture in DMEM on a 96-well U-bottom plate (late culture control), and day 30 of culture in chondrogenic differentiation media (differentiated chondrocytes). The cells directed for RNA isolation were collected from cultures with medium using a serological pipette and transferred into 1mL of the TRIzol reagent (Thermo-Fischer Scientific, Waltham, MA, USA). Then, the samples were stored frozen at −80 °C. RNA isolation was conducted with the use of the TRIzol Plus Purification KIT (12183555, Thermo-Fischer Scientific, Waltham, MA, USA), as described in the manufacturer’s protocol. The evaluation of the total amount of collected RNA was conducted using optical density at 260 nm, while its purity was determined based on the 260/280 nm absorption ratio (NanoDrop 2000 spectrophotometer, Thermo-Fischer Scientific, Waltham, MA, USA). Further studies were only based on samples a 260/280 absorption ratio higher than 1.8 and RNA content over 1 mg.