Rrna removal kit

After being obtained from surgical specimens, samples were immediately frozen using liquid nitrogen. Sample preparation and microarray hybridization were performed according to the protocols of Arraystar (Rockville, MD, USA). CircRNAs were enriched through removing linear RNAs with Rnase R (Epicentre, Madison, WI, USA), and then amplified and labelled using Arraystar Super RNA Labeling Kit (Arraystar). mRNAs were purified using rRNA removal kit (Arraystar). Subsequently, Arraystar Human circRNA Array (8 × 15K) and LncPath Human Cancer Array were used for hybridization, and then scanned by the Agilent Scanner G2505C (Jamul, CA, USA). circRNAs and mRNAs demonstrating fold-changes of ≥ 2 and p-values of less than 0.05 were regarded as significantly differentially expressed.

Keloid and normal skin specimens from 3 randomly selected patients were tested by microarray profiling. The chip used was the lncPath Human EMT Array (8 × 15 K) (ArrayStar, Rockville, MD, USA). Microarray analysis was performed by Kangchen Biology Engineering Co., Ltd., (Shanghai, China) according to the manufacturer’s protocol (Arraystar). LncRNAs/mRNAs were purified from total RNA after removal of rRNA (Arraystar rRNA removal kit). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen, Germany). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. One microgram of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl 25 × Fragmentation Buffer and then heating the mixture at 60 °C for 30 min. Finally, 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. Fifty microliters of hybridization solution was dispensed into the gasket slide and assembled into the lncRNA expression microarray slide. The slides were incubated for 17 h at 65 °C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent Scanner G2505C.

After extracting the total RNA, mRNA, and noncoding RNAs were enriched by removing rRNA from the total RNA with kit (Arraystar rRNA Removal Kit). The mRNAs and noncoding RNAs were fragmented into short fragments (about 200–500 nt) using the fragmentation buffer. First-strand cDNA was synthesized by random hexamer primer using the fragments as templates. During second strand synthesis, dTTP was substituted by dUTP. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. The purified short fragments were connected with adapters, then the second strand was degraded using UNG (uracil-N-glycosylase) [86 (link)]. After agarose gel electrophoresis, the suitable fragments were selected as templates for PCR amplification. An Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used for quantification and qualification of the sample library. The library was sequenced using a Illumina HiSeq^TM 2000 system.