key: cord-1043458-gbmyjnjg
authors: Mote, Ridim D; Laxmikant V, Shinde; Singh, Surya Bansi; Tiwari, Mahak; Hemant,; Srivastava, Juhi; Tripathi, Vidisha; Seshadri, Vasudevan; Majumdar, Amitabha; Subramanyam, Deepa
title: A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR
date: 2021-07-14
journal: bioRxiv
DOI: 10.1101/2021.07.14.452300
sha: 460fe5d950aabc5c6585bf0379337271f7a1b9db
doc_id: 1043458
cord_uid: gbmyjnjg

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

Real-time polymerase chain reaction (Real-time PCR) is a powerful technique to measure the level of gene expression. It is a quantitative PCR technique where data is collected simultaneously as the PCR amplification proceeds. It is an extremely sensitive technique with a large dynamic range and high sequence specificity (Wong and Medrano 2005) , with ability to even detect a single copy of a specific transcript (Palmer et al. 2003) . Real-time PCR is characterized by a Ct (Cycle threshold) value which indicates the cycle where the fluorescence intensity of the PCR product is greater than the background fluorescence (Heid et al. 1996) . Detection of the amplicon in real-time PCR can be done in multiple ways. The amplicon can be detected by using hybridization probes such as Taqman probes (Holland et al. 1991) , molecular beacons (Tyagi and Kramer 1996) , Eclipse Probes (Lukhtanov et al. 2007) , LUX PCR Primers (Vilcek et al. 2010) and Scorpions (Whitcombe et al. 1999) . While these probes are target sequence-specific, they are not commonly used due to their high cost. For the detection of a large number of genes, fluorescent DNA binding dyes, which are not sequence-specific, and intercalate between doublestranded DNA can be employed. Various DNA binding dyes such as SYBR Green I (Green and Sambrook 2018) and SYTO dyes (Gudnason et al. 2007; Eischeid 2011) are commercially used to detect the amplified PCR product, of which SYBR Green I is the most widely used. However, the commercially available SYBR Green I mastermixes are expensive. Hence, we set out with a goal to design a low-cost, inhouse, real-time PCR mastermix using reagents easily available in labs, and which are as efficient as commercially available mastermixes. dissociation curves were comparable between commercially available SYBR Green I PCR mastermix, and our in-house real-time PCR mastermix with either SYBR Green I or Evagreen dye. The in-house PCR mastermix is both sensitive, cost-effective and can be easily assembled. Our results therefore provide an effective solution towards developing an in-house real-time PCR mastermix which can be used as a costeffective and efficient alternative towards commercially available real-time PCR mastermixes.

Real-time PCR technique is an integration of 3 processes. RNA isolation, cDNA synthesis and real-time PCR (Fig. 1A) . One of the essential steps after RNA isolation is cDNA synthesis. There are a large number of commercially available kits for cDNA synthesis which are very expensive. Here, we have used a protocol for synthesis (Graham et al. 2021) ). cDNA synthesis was carried using 1µg of RNA from mouse ESCs as described in materials and methods. The efficiency of cDNA synthesis was analysed by GAPDH PCR (Supp. Fig. 1A ). Next, cDNA was diluted to 1:10 v/v and used as template for real-time PCR. Hot-start Taq polymerase was purified as per the previously published protocol (Graham et al. 2021) . Briefly, expression plasmid pET-28a_6H-TAQ_E602D (Addgene #166944) was transformed into BL21 competent cells, cultures were grown overnight at 37°C, induced with 1 mM IPTG, pelleted down and flash-frozen in liquid nitrogen and stored at -80°C until further use. Pellets were resuspended in lysis buffer and subjected to Ni-NTA based purification followed by HiTrap heparin purification.

Proteins fractions were eluted in storage buffer and stored at -80°C until further use (for buffer composition refer materials and methods and for detailed purification protocol refer (Graham et al. 2021) ). This hot-start Taq polymerase was used in the preparation of the in-house PCR mastermix ( Fig. 1D Table 5 . In summary, we demonstrate a cost-effective and efficient method to assemble mastermixes for cDNA synthesis and RT-PCR.

Real-time PCR is the most favoured technique for measuring gene expression. Here, one can measure the expression of several genes with relatively low amounts of sample. However, commercially available RNA isolation kits, cDNA synthesis kits and SYBR Green I mastermix required to set up real-time PCR are very expensive and cannot be afforded by several educational institutions and small enterprises. The ongoing coronavirus pandemic has negatively impacted the research funding in a large number of developing countries, including India. This adds to the additional hurdle which limits research scholars from using expensive cutting edge techniques such as real-time PCR. Earlier, there have been attempts to prepare homemade SYBR Green I mastermix for real-time PCR. However, this mastermix contained commercial Taq polymerase and cDNA was also synthesised using commercially available kits (Karsai et al. 2002) . This increases the expenses of conducting realtime PCR. While Graham et al demonstrate the assembly of home-made mastermixes for cDNA synthesis and real-time PCR, they do not provide a direct comparison with commercially available reagents for the same (Graham et al. 2021).

Our study provides such a direct comparison, and also includes alternatives to SYBR Green I, such as EvaGreen. Through our manuscript, we have systematically integrated and validated protocols for RNA isolation, cDNA synthesis and real-time PCR using in-house reagents. We have compared the efficacy of these reagents with commercially available kits. Our results demonstrate that real-time PCR set up using in-house SYBR Green I or EvaGreen PCR mastermix generated Ct values, amplification plots and dissociation curves comparable to commercially available SYBR Green I PCR mastermix. These in-house reagents are not only cost-effective, but are also sensitive. Hence, these in-house reagents for real-time PCR are a promising alternative to commercially available kits.

RNA isolation: Total RNA was isolated from mESCs using TRIzol (Invitrogen Cat no. 15596018). Culture media was removed and cells were washed once with 1XDPBS. 500µl TRIzol was added to lyse the cells and plates were kept on the rocker for 5 minutes. Cells were scraped and the lysate was collected in an eppendorf. 100µl of chloroform was added to the TRIzol lysate and mixed thoroughly by shaking. Samples were kept at room temperature for 5 minutes and then centrifuged for 15 minutes at 12000 × g at 4°C. The upper aqueous phase containing RNA was collected in a fresh eppendorf tube and 250µl isopropanol was added to the sample. Sample was mixed and kept at room temperature for 10 minutes and later centrifuged for 10 minutes at 12000 × g at 4°C. Supernatant was discarded and the pellet was washed with 70% ethanol. Sample was centrifuged for 5 minutes at 7500 × g at 4°C. The pellet was air dried and RNase free water or DEPC-treated water was added to the sample. RNA was quantified using Nanodrop spectrophotometer.

DNase treatment: 1ug of total RNA was used for DNAseI treatment (Invitrogen cat no. 18068-015). Details of the reaction are given below. Total reaction volume -20µl

The above reaction was assembled in PCR tubes and was placed in Eppendorf mastercycler PCR machine using the following program. DMIX10_100ML) were used to prepare mastermix. ABI 384 well plate (cat no. AB 1384) was used to set up real time PCR. 5µl of mastermix was added to each well.

1µl of (1:10) diluted cDNA was added as template. Total reaction volume was 6µl.

ABI 7900 HT machine was used to perform Real-time PCR. Detailed composition of SYBR Green I and EvaGreen PCR mastermix is given below.

2X In-house buffer -1000µl 2X In-house buffer -1000µl 10mM dNTP -25µl 10mM dNTP -25µl 10000X SYBR Green I -0.1µl 20X Evagreen -100µl Hotstart Taq -10 µl Hotstart Taq -10µl Both, 2X In-house SYBR Green I mix and 2X In-house EvaGreen mix can be stored at 4°C for up to 2 weeks. For long term storage, mastermix was stored at -20°C.

2X In-house SYBR Green I mix or 2X In-house EvaGreen mix -3µl 100µM Forward primer -0.6µl 100µM Reverse primer -0.6µl Milli Q water -1.88µl

Total reaction volume -6µl

Stage 1 50°C -2 minutes Supplemental Table 1 : Ct values for pluripotency marker genes Oct4, Sox2, Nestin, Brachyury and housekeeping genes Gapdh and Rpl7 in mESCs with different concentrations of dNTPs 250µM, 300µM and 400µM using in-house EvaGreen PCR mastermix. S1 -Sample 1 and S2 -Sample 2. Pipetting duplicates for each sample are shown.

Supplemental Table 2 : Ct values for pluripotency marker genes Oct4, Sox2, Nanog and housekeeping genes Gapdh and Rpl7 in mESCs with commercial SYBR Green I mastermix.

Supplemental Table 3 : Ct values for pluripotency marker genes Oct4, Sox2, Nanog and housekeeping genes Gapdh and Rpl7 in mESCs with in-house SYBR Green I mastermix.

Supplemental Table 4 : Ct values for pluripotency marker genes Oct4, Sox2, Nanog and housekeeping genes Gapdh and Rpl7 in mESCs with in-house EvaGreen PCR mastermix. 

acknowledge intramural funding from National Centre for Cell Science. S.B.S is a recipient of a Senior Research Fellowship from the Department of Biotechnology, India; M.T. and J.S. are recipients of a Senior Research Fellowship from University Grants Commission, India; H. is a recipient of a Senior Research Fellowship from CSIR, India. We thank members of the Subramanyam and Majumdar lab for constructive discussion

M. conceived and designed the study. R.D.M. contributed to experiments in Fig. 1A, 2, 3, 4, Fig. S1, Table S1-S4

This work was supported by funds to D.S. from the Department of Biotechnology

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