Cdna ecodry premix

The expression of ERV elements was measured using qPCR as described previously [48 (link)]. GAPDH mRNA expression served as the internal control. The assay was conducted using Bioneer SYBR Green qPCR Premix and an Applied Biosystems 7500 PCR instrument. Total RNA was isolated with TRIzol reagent, and 2.5 ng total RNA was used to produce cDNA from the isolated RNA with cDNA EcoDry Premix (Takara, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions. The primer sequences used in this study are listed in Additional file 1: Table S2.

The retina and RPE/choroid were dissected from at least three non-diseased human eyes of similar age (male and female) and cut into smaller pieces in cold PBS. The tissue samples were snap frozen in liquid nitrogen and stored at –80ºC for RNA preparation. Tissue samples (50–100 mg) were dissolved in 1 mL of Trizol reagent (Invitrogen, San Diego, CA). Total RNA was extracted using RNeasy mini kit as recommended (Qiagen, Valencia, CA). Complementary DNA was prepared using 1 μg of total RNA and the RNA to cDNA EcoDry Premix (TaKaRa, Mountain View, CA) and diluted 1:10. qPCR was conducted in triplicates using a Mastercycler Realplex (Eppendorf, Enfield, CT) and TB-Green qPCR Premix (TaKaRa). The cycles for amplification were 95ºC for 2 min; 40 cycles of amplification (95ºC for 15 s, 60ºC for 40 s); and dissociation curve step (95ºC for 15 s, 60ºC for 15 s, 95ºC for 15 s). The relative fluorescent units (RFUs) at a threshold fluorescence value (Ct) were used for linear regression line and assessment of nanograms of DNA. The target gene expression levels were determined by comparing the RFU at the Ct to the standard curve and normalized by the housekeeping gene ribosomal protein L13α (RPL13A). The primer sequences used in this study are listed in Table 1. Each sample was run in triplicates.

RNA was extracted from rat maxillae frozen from 4 and 8 weeks groups and from the human periodontal ligament progenitors using RNeasy Plus kit (Qiagen, Hilden, Germany). For the RNA extraction from rat maxillae, the samples were first frozen using liquid nitrogen and immediately ground into powder form using a mortar pestle before proceeding to use the RNA extraction kit. One microgram of total extracted RNA was applied towards cDNA generation with RNA to cDNA ecodry premix (Takara, kusatsu, Japan). Real-time RT-PCR was performed using the SYBR green master mix, and sequence specific primers were designed for the study (Table 1) and CFX96 Real Time system (Bio-Rad). The reaction conditions were as follows: 2 min at 50 °C (1 cycle), 10 min at 95 °C (1 cycle), and 15 s at 95 °C, and 1 min at 60 °C (40 cycles). Samples were normalized against GAPDH. To quantify the relative differences in mRNA expression, the standardized comparative CT method (ΔΔCT) was used to determine relative quantity. All values were graphed as the relative mean expression level ± standard deviation. The gene expression for the in vivo rat samples was confirmed for Collagen I (Col I), Collagen III (Col III), E-cadherin (Cdh1), Fibronectin (Fn), Vegf, Runx2 and Osx for the rat samples. For the in vitro samples, gene expression was confirmed for COL I, COL III, FN, VINCULIN (VCL), and VEGF.