Fast 96 well reaction plate

Total RNA was isolated using Isogen reagent (Nippon Gene, Toyama, Japan) in accordance with the manufacturer’s instructions. cDNA copies of the total RNA were synthesized using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). The reaction mixture included 9 µL samples of total RNA, 10.0 µL of 2 × RT buffer, and 1.0 µL of 20 × RT enzyme mix. Conditions for the reverse transcription were as follows: 37 °C for 60 min and then 95 °C for 5 min. A total of 20.0 µL of reaction mixture containing 10.0 µL of TaqMan gene expression master mix (2 ×), 1.0 µL of TaqMan gene expression assay (20 ×), 4 µL of cDNA, and 5 µL of H₂O were added to each well of a Fast 96-Well Reaction Plate (0.1 mL, Applied Biosystems). Quantitative RT-PCR was performed using primers for Insulin 1 (Ins1: TaqMan assay ID: Rn0212433_g1), Insulin 2 (Ins2: TaqMan assay ID: Rn01774648_g1), and amylase (Amy 2a3: TaqMan assay ID: Rn00821330-g1) on a StepOnePlus real-time PCR system (Applied Biosystems). The threshold cycle was calculated automatically using the instrument software, with a threshold value of 0.2. For this study, β-actin (TaqMan assay ID: Rn00667869_m1) was used as the endogenous reference gene. TaqMan Gene Expression Assays were purchased from Applied Biosystems and tested in accordance with the manufacturer’s protocol [42 (link)].

Potential DNA contamination from RNA samples was removed with DNAseI (Fermentas, Burlington, Ontario, Canada). Complementary DNA (cDNA) synthesis was then performed using Maxima Reverse Transcriptase (Fermentas). After addition of the mix containing reverse transcriptase, an RNAse inhibitor, random hexamer, and a solution with the 4 deoxynulceotide triphosphates, samples were incubated in a thermal cycler (BioRad My Cycler) as follows: 10 min at 25°C, 30 min at 50°C to achieve full polymerase activity and 5 min at 85°C to inactivate the enzyme.
For quantitative real-time PCR samples were assayed in Fast 96-well reaction plate (Applied Biosystems, Foster City, CA, USA) with each condition containing 100 ng of cDNA in a total volume of 2.5 μL sterile water with 0.3 μM of each forward and reverse primer (Integrated DNA Technologies, Coralville, IA), 5 μl iTAQ SYBR Green Supermix with Rox (BioRad) as well as 1.9 μl sterile water. Quantitative real-time PCR (qRT-PCR) was carried out in a Step-One-Plus machine (Applied Biosystems, Foster City, CA). Each condition was normalized to the housekeeping gene GAPDH. Relative fluorescence was interpreted as fold induction from cycle threshold values using the Pfaffl mathematical model.
Primer sequences are:

Total RNA was extracted from cells and 1 μg was used to synthesize cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) after 3, 7, 14, and 21 days. ALP, Runx2, BMP, and OPN mRNA expression was investigated by qRT-PCR on a StepOne Plus Real-Time RT-PCR system (Applied Biosystems). Taqman Fast Universal PCR Master Mix (10 μL), 1 μl of the primer probe set (20× Taqman Gene Expression Assays), sample cDNA (2 μL), and diethylpyrocarbonate-treated water (Nippongene, Toyama, Japan; 7 μL) were added to each well of a Fast 96-well Reaction Plate (0.1-mL well volume; Applied Biosystems). The plate was subjected to 40 reaction cycles of 95 °C for 1 s and 60 °C for 20 s. Target gene expression level was calculated with the 2^−ΔΔCt method relative to the negative control group.