Miscript universal primer from the miscript sybr green pcr kit

Quantitative RT-PCR was carried out in technical duplicates using the QuantiTect SYBR Green PCR Kit (Qiagen) according to the manufacturer’s two-step RT-PCR protocol. To measure miR-4649-5p expression, the Hs_miR-4649-5p_1 miScript Primer Assay (Qiagen) was used together with the miScript Universal Primer from the miScript SYBR Green PCR Kit (Qiagen) and was normalized to the two housekeepers SNORD61 and SNORD95 using the Hs_SNORD61_11 and Hs_SNORD95_11 miScript Primer Assays (Qiagen) according to the manufacturer`s instructions.
For the detection of coding genes together with the housekeepers GAPDH and U6, primers were designed with the NIH Primer Blast Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and purchased from Eurofins Scientific. A list of these primer sequences is given in Additional file 1: Table S1.
Per qPCR reaction (volume of 10 µl), 1 ng of cDNA was used. The measurements were carried out in LightCycler® 480 Multiwell Plates 384 on a LightCycler® 480 Real-Time PCR System (Roche, Basel, Switzerland). Ct values were normalized by subtracting the respective arithmetic mean of the two housekeeper genes to receive delta Ct (ΔCt) values. Following the ΔΔCt method, relative expression levels were calculated by subtracting the ΔCt of the respective negative control and were plotted as 2^−ΔΔCt.

RT-qPCR was performed in technical duplicates with the QuantiTect SYBR Green PCR Kit (Qiagen) following the manufacturer’s two-step RT-PCR protocol. The following primers were used:
The Hs_miR-4646-5p_1 miScript Primer Assay (Qiagen) together with the miScript Universal Primer from the miScript SYBR Green PCR Kit (Qiagen). MiR-4646-5p expression was normalized to the two housekeepers SNORD61 and SNORD95 using the Hs_SNORD61_11 and Hs_SNORD95_11 miScript Primer Assays (Qiagen) according to the manufacturer`s instructions.
Primers for the detection of coding genes and the two corresponding housekeepers GAPDH and U6 were designed with the NIH Primer Blast Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and ordered from Eurofins Scientific (Luxembourg). A list of these primer sequences is given in Supplementary Table S1.
Per RT-qPCR reaction, 1 ng of cDNA was applied in a 10 µL reaction volume. Measurements were conducted in LightCycler^® 480 Multiwell Plates 384 on a LightCycler^® 480 Real-Time PCR System (Roche, Basel, Switzerland). For Ct value normalization, the arithmetic means of the according housekeeper genes were subtracted to receive delta Ct (ΔCt) values. Relative expression levels were calculated by subtracting the ΔCt of the respective negative control and were plotted as 2^−ΔΔCt.