Highly sensitive and multiplexed quantification of mRNA splice variants by the direct ligation of DNA probes at the exon junction and universal PCR amplification

A highly sensitive and specific assay for detecting mRNA splice variants is developed based on ligation-dependent PCR.

All the reagents were of analytical grade and were used as received without further purification.
The sequences of the oligonucleotides were given in the Table S1.
The total RNA extracts were stored in Forma ultra-low temperature freezer (Thermo Scientific, USA). Temperature control device was 2720 PCR system (Applied Biosystems, USA). DYY-6D electrophoresis apparatus (Beijing Liuyi Biotechnology, China) and Gel DoxTM EZ Gel imaging system (BIO-RAD, USA) were used to perform electrophoresis separation and imaging analysis of PCR products. NanoDrop 2000 (Thermo Scientific, USA) was used to quantify the total RNA extracts. The real-time fluorescence measurements were performed with the StepOne Real-Time PCR system (Applied Biosystems, USA). Reverse Primer CCATCTCATCCCTGCGTGTC * The letter "r" indicates "ribonucleotide".

Preparation of total RNA extracts
The MCF-7, Hela, HCT-116 and MRC-5 cell lines were cultured in 6 mL DMEM Medium (GBICO, Cat. 12100-046) containing 10% (v/v) fetal calf serum (GBICO, Cat. 1600036), 1% NaHCO 3 , 100 U/mL penicillin, 100 μg/mL streptomycin and 3 mmol/L L-glutamine. All of the cell lines were maintained at 37 °C under a humidified atmosphere containing 5% CO 2 . Total RNA was extracted respectively from the MCF-7, Hela, HCT-116 and MRC-5 cell lines using a TRIzol ® Reagent (Invitrogen, USA) according to the manufacture′s protocol.  μL RNase-free water with final volume 10 μL. PCR reaction were carried out with 2720 PCR system by using hot start of 94 °C for 2 min, followed by 25 cycles of 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 20 s. Different mRNA splice variants were detected simultaneously by separating amplification products using non-denaturing polyacrylamide gel electrophoresis. The gel was stained by 4S Red Plus Nucleic Acid Stain and visualized using a Gel Doc EZ Imager (Bio-Rad, USA).

Specificity comparison between RT-PCR and the direct ligation-based PCR
As shown as in Fig. S1

Optimization of PCR cycle number for multiplex mRNA splice variants assay
PCR cycle numbers determine how much the PCR amplification products, and the electrophoresis detection need enough PCR amplification products to accomplish imaging analysis. So the PCR cycle number was an important parameter for ligation-depended PCR assay to detect different mRNA splice variants simultaneously. We chose three synthetic Δ (11q, 3642-), Δ (11q, 333-), and Δ (9, 10) as the model to optimize PCR cycle number for the multiplexed analysis of mRNA splice variants. The optimal PCR cycle number was investigated by detection the mixture including Δ (11q, 3642-), Δ (11q, 333-) and Δ (9, 10) at 0, 1 fM, 10 fM and 100 fM, respectively.
After 23, 25 and 30 cycles, PCR amplification products were characterized by gel electrophoresis.
As shown in Fig. S2, with the increasing the PCR cycle numbers, the grayscale of the products  products shows a defined band corresponding to 97 bp, Δ (9, 10) products shows a defined band corresponding to 114 bp.

Optimization of T4 RNA ligase 2 dosage for multiplex mRNA splice variants assay
Ligase dosage not only affect the efficiency of ligation reaction but also the specificity of the ligation reaction. Therefore, T4 RNA ligase 2 dosage was investigated by testing Δ (11q, 3642-), Δ (11q, 333-), and Δ (9, 10) at 0, 1 fM, and 10 fM, respectively with the ligation-depended PCR assay. 0.05 U/μL, 0.1 U/μL, and 0.2 U/μL T4 RNA ligase 2 were investigated, as shown in Fig.   S3. When T4 RNA ligase 2 dosage was 0.05 U/μL, 10 fM mRNA splice variants was detectable, and the bands of non-specific amplification products(lane 2) and amplification products of 1 fM mRNA splice variants (lane 3) can not seen. This is due to ligase dosage was too low, nonspecific S7 connections decreased, while specific connection between the probes and mRNA splice variants also decreased. Thus, the amount of the PCR amplified template was reduced, which did not produce enough amplification products to eletrophoresis separation. When T4 RNA ligase 2 dosage was 0.1 U/μL, the bands of non-specific amplification products(lane 5) and amplification products of 1 fM mRNA splice variants (lane 6) can be seen, and the bands grayscale of lane 6 were deeper than lane 5. That is say that 1 fM mRNA splice variants was detectable using 0.1 U/μL T4 RNA ligase 2. When T4 RNA ligase 2 dosage was 0.2 U/μL, the bands of non-specific amplification products(lane 8) and amplification products of 1 fM mRNA splice variants (lane 9) can also be seen, but the bands grayscale was different to distinguish between lane 8 and lane 9.
Therefore, 0.1 U/μL was selected as the optimum T4 RNA ligase 2 dosage for multiplex alternative splicing assay.