Miscript universal primer

sRNAs were extracted from mushroom caps and stipes using miRNeasy Serum/Plasma Kit (Qiagen, Madrid, Spain) according to the manufacturer’s protocol and quantified with a NanoDrop2000 (Thermofisher, Madrid, Spain). Retrotranscription (RT) was carried out using an miScript^® II RT (Qiagen, Madrid, Spain) kit, then the real-time PCR was used on a 7900HT Fast PCR (Applied Biosystems, Madrid, Spain) selecting 15 min at 95 °C, then 40 cycles of 15 s at 94 °C, 30 s at 50 °C and 45 s at 70 °C. Samples were mixed with the SYBR Green (Qiagen, Madrid, Spain) as fluorochrome. miScript Universal Primer (Qiagen, Madrid, Spain) for miRNAs and primers listed in Table S5 was used in this study. Their consensus mature sequence (CMS), consensus star sequence (CSS) and consensus precursor sequence (CPS) were evaluated using rRNA 5.8S as housekeeping RNA expression with the SYBR Green real-time PCR method. A no template control was used as negative control of real-time PCR. This negative control omits any DNA or RNA template from the reaction and serves as a general control for nucleic acid contamination or as control for primer dimer formation. An ANOVA and pair-wise Bonferroni tests were performed with R to discriminate differences between means corresponding to different samples.

The StepOne^™ Real-Time PCR kit from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the cDNA copy number. Amplifications were performed using the miScript SYBR Green PCR kit (Qiagen GmbH) with a reaction mixture consisting of 2.5 μl 10X miScript Universal Primer, 12.5 μl 2X QuantiTect SYBR Green PCR Master Mix, 5 μl RNase-free water, 2.5 μl cDNA, and 2.5 μl specific primers for MiRNA-567. Initial denaturation was performed at 95°C for 15 min, then at 94°C for 15 sec, 55°C for 30 sec, and 70°C for 30 sec for 40 cycles. The melting curve was attained from 65°C to 95°C (S3a and S3b Fig). A quantitation cycle (Cq) value < 30 was an acceptable amplification and a Cq value > 35 was considered unacceptable. The qPCR primers are shown in Table 2. miRNA-282, miRNA-989 (available from the commercial kit, MBI Fermentas, Germany), and RNU6B primer were applied according to the manufacturer’s instructions and Morin et al. [38 (link)]. Each sample was analysed in duplicate. The relative expression of the miRNA was calculated using the 2^−ΔΔCT method.

For real-time PCR we used miRNA-499 and miRNA-208 miScript Primer Assays and the miScript SYBR Green PCR Kit, which contains the miScript Universal Primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix (Qiagen, Germany).
PCR was performed by initial activation step for 15 min at 95 °C, and 3-step cycling, including denaturation step for 15 s at 94 °C, annealing step for 30 s at 55 °C and extension step for 30 s at 70 °C. The total number of cycles was 40.
To analyze the cDNA content of various microRNAs in each sample, we used the values of Ct (test) and Ct (ref), where Ct (ref) is the intersection point of the baseline and the amplification graph of the reference gene (RNU-6) in the sample, and Ct (test) is the intersection point of the baseline and a graph of amplification of the studied gene in the same sample.

cDNA from oocyte and embryo samples (n = 3 pools of five each) was prepared by lysing the samples in 1× miScript RT buffer containing 0.5% NP-40 at 95 °C for 5 min followed by addition of miScript reverse transcriptase mix (Qiagen, Valencia, CA) and incubation at 37 °C for 60 min. The cDNA was then used for determination of relative amount of miR-1296 by RT-qPCR using the miRNA-1296 specific primer and the miScript universal primer (Qiagen, Valencia, CA). Bovine miRNA-125b was used as an endogenous control as this miRNA is expressed consistently in preimplantation embryos [46 (link)]. RT-qPCR analysis was performed on the Bio-Rad CFX96 system. The iQ™ SYBR Green Supermix (Bio-Rad, Hercules, CA) was used in 20 μl reaction volumes containing 100 nM of each primer and 5 μl of diluted cDNA. Cycling parameters were 95 °C for 15 min, and then 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Standard curves for the target and control miRNA were constructed using 10-fold serial dilution of a pooled cDNA sample.

On the basis of results obtained from the array, we studied miRNAs up-regulated or selectively expressed by patients in a cohort of additional 16 OSCC patients and 6 controls. 500 ng of total RNA was retro-transcribed to cDNA with miScript II RT Kit (Qiagen, Hilden, D) and evaluated for the expression of five miRNAs up-regulated in patients compared to healthy controls (miR-412-3p, miR-489-3p, miR-512-3p, miR-597-5p, and miR-603). Furthermore, eight miRNAs exclusively expressed only in OSCC patients (miR-27a-3p, miR-302b-3p, miR-337-5p, miR-373-3p, miR-494-3p, miR-517b, and miR-520d-3p, miR-645) were also evaluated. Each sample was run in triplicate and each miRNA-specific primer was run in a separate reaction. SnoRNA RNU6B and miR-191 were used as endogenous control as previously described [18 (link)–20 (link)] due to their stable expression in saliva samples which was also confirmed in the current study by qRT-PCR in tested salivary EV samples.
The qRT-PCR reaction mix was composed of 2 ng of cDNA, 100 nM miScript Universal Primer (Qiagen), 100 nM miRNA-specific primer (Eurofins Genomics, Ebersberg, D), 5 μl QuantiTect SYBR Green PCR Master Mix (Qiagen), and nuclease free water (Qiagen) to reach a final reaction volume of 10 μl. The Real-Time Thermal Cycler Quant Studio 12 k (Thermo Fisher Scientific) was used for analysis.

The levels of miR-210 were detected as we previously described [32 (link), 33 ]. Total RNA was extracted using the TRIzol reagent (15596026; Invitrogen). MiR-210 levels were analyzed by miScript II RT kit (218161, Qiagen) and miScript SYBR Green PCR kit (218073, Qiagen) with miScript Primer Assay kit (MS00000644, Qiagen) according to the manufacturer’s instructions. Briefly, 1 μg of template RNA was mixed with reverse-transcription master mix in a final volume of 20 μl and incubated at 37 °C for 60 min, and then the reaction was stopped at 95 °C. Two nanograms of template cDNA was used for miR-210 quantification in a final volume of 25 μl system containing specific primers and QuantiTect SYBR Green PCR master mix according to the manufacturer’s instructions. Primers included miScript Universal Primer, miR-210 miScript Primer Assay, and SNORD61 miScript Primer Assay (MS00033705, Qiagen). PCR was done in triplicate and threshold cycle numbers were averaged for each sample. The relative expression levels of mature miR-210 were calculated using the formula 2(^-ΔΔCt) and normalized to SNORD61. The change of miR-210 was expressed as fold of normal control.