Trizol lysis reagent

Total RNA was isolated using TRIzol Lysis Reagent (Invitrogen), and cDNA was synthesized by All-in-OneTMFirst-Strand cDNA Synthesis Kit (GeneCopoeia, USA). For qPCR, amplification reactions were performed using All-in-One™qPCR Mix (GeneCopoeia, USA), run on the ABI-Ⅶ Real-Time PCR detection system. The primers were synthesized by Tsingke Company (China); the details of sequences are listed in Supplementary Table S3. All experimental steps were performed following the manufacturers’ instructions. Data analysis was performed using the 2^−(ΔΔCT) method to determine the relative quantitative level and was expressed as a fold-difference to the relevant control (recognized as 1 ± 0.00).

For qPCR analysis, larvae were collected (15 per sample) in TRIzol lysis reagent (Invitrogen) for RNA isolation, which was performed using the miRNeasy mini kit (Qiagen, Venlo, The Netherlands), according to the manufacturer’s instructions. For cDNA synthesis, the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) was employed, using 1 µg of each RNA sample. Subsequently, 17 genes were analyzed using qPCR using the MyiQ single-color real-time PCR detection system (BioRad Laboratories). The analysis was performed in a total volume of 25 µL containing 5 µL cDNA, 5.5 µL water, 1 µL forward and 1 µL reverse primer (10µM), and 12.5 µL of 2× iQ SYBR Green Supermix (Bio-Rad Laboratories). PCR settings were: 95 °C for 3 min, 40 cycles of 15 s at 95.5 °C, 15 s at 60 °C, and 30 s at 72 °C. Cycle threshold (Ct) values were defined for each run. For every sample, The Ct value was deducted from the Ct value of a control sample. The fold alteration of gene expression was calculated and normalized to the expression levels of peptidylprolyl isomerase Ab (ppial) as a reference gene. The lid temperature was 105 °C. An additional protocol was included to determine the dissociation of the PCR products from 65 °C to 95 °C, permitting the recognition of the amplified products. Reactions were performed in duplicate. The sequences of all qPCR primers are presented in Table S1.

Total RNA from cells and homogenized lung tissue was extracted using TRIzol lysis reagent (15596026; Invitrogen, Waltham, MA, USA) and reverse transcribed using the SuperScript^® III First-Strand Synthesis System for RT-PCR (18080-051, Invitrogen) per manufacturer’s instructions. Quantitative real-time PCR (RT-PCR) was performed using primer-probes for human MMP-9 (Hs00957562_m1, Thermo Fisher) and mouse MMP-9 per manufacturer’s instructions (Mm00442991_m1, Thermo Fisher). RT-PCR was performed on the MyiQ™ Single-Color Real-Time PCR detection System (Bio-Rad, Hercules, CA, USA) using SYBR Green PCR Master Mix per manufacturer’s instructions (4309155; Applied Biosystems, Waltham, MA, USA). RT-PCR was performed using an initial 10 min denaturation period at 95 °C followed by 50 cycles of 15 s at 95 °C and 1 min annealing and extension at 60 °C. Expression levels of MMP-9 were normalized to 18S RNA.

Breast tissue specimens or cell pellets were homogenised in 1 ml TRIzol® lysis reagent (Invitrogen) as previously described [27 (link)]. Total (large and micro) RNA was extracted from malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) mammary tissue using the RNeasy Mini Kit (QIAGEN) as per manufacturer’s instructions.

Total mRNA was extracted from 1.0 × 10⁶ cells using TRIzol Lysis Reagent (Invitrogen, Thermo Fisher Scientific, Milan, Italy, Cat. 15596026) according to the manufacturer’s instruction. The first strand of cDNA was synthesized from 0.5–1 µg of total RNA using Im-Prom-II system (Promega, Madison, WI, USA, Cat. A3800). Real-Time PCR was performed using iTaq qPCR master mix, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA, Cat. 1725124), in a SFX96 real-time system (Bio-Rad, Hercules, CA, USA). To normalize raw real-time PCR data, an S18 ribosomal subunit was used. Primers used were listed in Table 3. Data are expressed as delta-C (t) of the gene of interest to S18, allowing appreciation of single gene expression levels.