Taqman gene expression master mix

To determine CTSB mRNA expression in cancer tissue a cDNA array (CSRT103, Origene, Rockville, MD, USA) was commercially obtained. Furthermore, mRNA of cancer cell lines was isolated using peqGOLD RNA pure (Axon Lab, Baden, Switzerland) and reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher). The amount of CTSB was assessed by quantitative real-time PCR (qPCR) using the pre-developed TaqMan^TM assays (Thermo Fischer) Hs00947439_m1, the TaqMan^® gene expression mastermix, and the ViiA^TM 7 Real-Time PCR System. The reaction was carried out in a volume of 15 µL composed of 0.75 µL of the Hs00947439_m1-FAM TaqMan assay, 6.75 µL H₂O, and 7.5 µL TaqMan^® Gene Expression Mastermix (Applied Biosystems, LubioSciences, Lucerne, Switzerland). For quantification of copy numbers, a standard curve using cloned PCR amplicon was recorded.

dPCR was performed on a BioMark platform (Fluidigm Corporation, San Francisco, California) with qdPCR37K integrated fluidic chips, following the manufacturer’s instructions with slight modifications. Instead of using 1.8 μL of DNA template, 2.1 μL of diluted pre-amplified DNA was used in each 6-μL PCR reaction. Patient samples underwent dPCR with each of the four TaqMan assays in a singleplex format using the TaqMan Gene Expression Master Mix (Life Technologies, Carlsbad, California) with a protocol of 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The presence of a single red spot with a sigmoidal amplification curve with a cycle threshold (Ct) value ≤23 in each panel was considered as a positive result.
qPCR was performed with 3 μL of diluted pre-amplified DNA as the template and the TaqMan Gene Expression Master Mix in a total volume of 20 μL on a QuantStudio 7 Flex Real Time PCR System (Life Technologies, Carlsbad, California), following the vendor’s instructions. The PCR parameters were the same as those described above. A Ct value <35 combined with a sigmoidal amplification curve was considered as a positive detection result. All qPCR reactions were performed in triplicate and repeated thrice to monitor reproducibility.

Cells were lysed in 1 mL of TriZol Reagent (Invitrogen), and the RNA was purified with the RNeasy Mini Kit (Qiagen). RNA was then reverse-transcribed with a High Capacity cDNA Reverse Transcripiton Kit (Applied Biosystems). TaqMan probes were designed with the TaqMan Gene Expression Assay tool (Applied Biosystems). The qRT-PCR was performed using a TaqMan^® Gene Expression Master Mix (Applied Biosystems) in a CFX384 TouchTM Real-Time PCR Detection System (Bio-Rad). Fold change in gene expression was estimated using the computed tomography comparative method and normalizing to the Gapdh computed tomography values and relative to control samples (Table 1).Table 1

Accession numbers for genes used in quantitative PCR

Gene	TaqMan® Gene Expression Master Mix (Applied Biosystems) accession number
Cyclin B1	Mm01322149_mH
Atoh1	Mm00476035_s1
Tuj1	Mm00727586_s1
Gap43	Mm00500404_m1
Gapdh	Mm99999915_g1
Brd4, exon 4-5	Mm01348074_m1
Brd4, exon 5-6	Mm00480392_m1
Brd4, exon 7-8	Mm00480394_m1
Brd2	Mm01271171_g1
Brd3	Mm00469733_m1
Gli1	Mm00494654_m1
Gli2	Mm01293117_m1
Gli3	Mm00492337_m1
Cyclin A1	Mm00432337_m1
Cyclin D1	Mm00432359_m1

Total mRNA was isolated from the mouse liver according to a TRIzol extraction protocol. After RNA quantification by Nanodrop (Thermo Scientific^®) and Qubit (Invitrogen^®), reverse transcription into cDNA was performed using a QuantiTect Reverse Transcription Kit (Qiagen^®) according to the manufacturer’s instructions. qRT-PCR was used to analyze the gene expression using a TaqMan Gene Expression MasterMix (Applied Biosystems^®). The reaction was performed using 96-well plates with a final volume of 5 µL, which included 1 µL of cDNA and 0.25 µL of each pre-manufactured gene sequence (PI3Kp85α-Mm00803160_m1; AKT1/2-Mm01331626_m1; GSK3β-Mm00444911_m1; glycogen synthase 2-Mm01267381_m1; G6Paseα-Mm00839363_m1; GLUT4-Mm01245502_m1; PEPCK1-Mm01247058_m1; glycogen phosphorylase-Mm01289790_m1; GAPDH-Mm03302249_g1) (TaqMan Gene Expression Assay, Applied Biosystems^®), 2.5 µL of the TaqMan Gene Expression MasterMix (Applied Biosystems^®) and 1.25 µL of RNase-free water. The reaction was performed in 40 cycles of StepOnePlus (Applied Biosystems^®). The data obtained were analyzed using the relative quantification method of gene expression (ΔΔCt). GAPDH was used as the endogenous control (Calegari et al. 2012 (link)).

qPCR was performed using TaqMan gene expression assays on the ABI PRISM 7900HT Sequence Detection System. The FAM-conjugated TaqMan probes were used along with the TaqMan Gene Expression Master Mix (Applied Biosystems, 4369510). Gene expression assays were normalized to endogenous VIC-conjugated Actb probes as standard. All RNA samples were immediately processed for complementary DNA preparation using the SuperScript IV First-Strand Synthesis System (Invitrogen, 18091200). To perform qPCR, FAM-conjugated TaqMan probes were used along with TaqMan Gene Expression Master Mix (Applied Biosystems, 4369510).