Mircury lnatm synthesis kit 2

To determine the expression of miRNAs (miR-21, miR-182, miRNA-7 and miR-SNORD 44) (Exiqon) in control non-treated cells/PRP treated cells, TRIZOL Reagent (Life Technologies) was used according to the manufacturer’s recommendations. Reverse transcription from miRs was performed using the miRCURY LNATM Synthesis kit II (Exiqon) following the manufacturer’s protocol. qPCR was performed using miRCURY LNA TM EXILENT SYBR Green (Exiqon) in a CFX96 real-time PCR detection system (Bio-Rad). Each reaction was performed in triplicate and the comparative threshold cycle (Ct) method was used to calculate the amplification factor. Human Snord 44 was used as housekeeping.

Total RNA from different cell lines was extracted from both 80% confluent adherent cell and ALDH1 + mammospheres after 5 days of culture in cell suspension, using the TRIZOL reagent following the manufacturer’s instructions (Sigma‐Aldrich). cDNA was synthesized by reverse transcription of total RNA using the Reverse Transcription System (Promega, Madison, WI, USA) for mRNA, and miRCURY LNA TM Synthesis kit II (Exiqon, Vedbaek, Denmark) for miRNAs. Quantitative real‐time‐PCR (qRT‐PCR) assay was done using SYBR Green PCR Master Mix (Promega) and miRCURY LNA TM EXILENT SYBR Green (Exiqon) for miRNAs. Each experiment was done in duplicate, and reactions were performed in triplicate. The comparative threshold cycle (Ct) method was used to calculate the amplification factor as specified by the manufacturer ABI 7500. For mRNAs, human GAPDH was used as an internal standard to normalize and hsa‐miR‐24‐3p, RNU6, and hsa‐miR‐425‐5p for miRNAs. The amount of target and endogenous reference was determined from a standard curve for each experimental sample. Primer sequences are listed in Table S1 (mRNAs) and Table S2 (miRNAs).

Total RNA, including miRs, was obtained from the different cell lines and subpopulations using the miRNeasy Mini Kit (Qiagen, Limburgo, Netherland) following manufacturer´s instructions. Reverse transcription from miRs was performed using the miRCURY LNATM Synthesis kit II (Exiqon, Vedbaek, Denmark) following manufacturer’s protocol. qPCR was performed using miRCURY LNA TM EXILENT SYBR Green (Exiqon) in a CFX96 real-time PCR detection system (Bio-Rad). Each reaction was performed in triplicate and the comparative threshold cycle (Ct) method was used to calculate the amplification factor. UniSp6 RNA Spike-in control primer set is used to amplify UniSp6 RNA Spike-in template for the control of the cDNA synthesis step. The standard curve was constructed by 5-fold serial dilutions of cDNA.