Complementary DNA synthesis

Complementary (cDNA) synthesis is an essential step in RNA-based BFID analysis. RNA must become cDNA in order to undergo PCR. In this protocol two different primers types were used. A standard reverse transcription method was used for large RNA transcripts e.g. mRNA (1500 to 2000 nt). In this method a combination of RT reagents including primers were used to synthesize a cDNA strand.

A stem-loop reverse transcription method was used for shorter RNA transcripts e.g. mature miRNA (18-25 nt). The shorter length of mature miRNA makes it difficult for primers to bind and accurately synthesize a cDNA strand. Either poly-T primers utilised after polyadenylation (SYBR® Green chemistry) or stem-loop primers (TaqMan® chemistry) can be used for this. In this work, stem-loop primers were selected due to their high specificity (e.g. of as little as one nucleotide difference) and use in forensic RNA research [244].

During RT, the 3‟ of the stem-loop primer will bind to the last 6 bp of the mature miRNA sequence while the 5‟ end, which is artificial (in nature) will fold on itself (shown in Figure 3). MMLV-RT will then extend this sequence. The stem-loop will unfold during PCR.

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Figure 2. A diagram showing cDNA synthesis using stem-loop primers. During reverse transcription, the 5’ end of the stem-loop primer will fold onto itself while the 3’ end of the stem-loop primer will bind to the last 6 bp in the mature miRNA sequence. The sequence is then extended using an enzyme e.g. MMLV-RT.

2.5.1 Standard reverse transcription

The RETROscript® first strand synthesis kit (Life Technologies, UK) was used for mRNA transcripts. Samples underwent standard reverse transcription following the RETROscript® protocol [245]. Samples were initially heat denatured with 50µM random decamers and nuclease free water on a Veriti thermocycler (Fisher Scientific, UK) for 3 min at 75 ºC to remove any secondary structure that may inhibit reverse transcription. A master mix containing the essential components for reverse transcription (2.5 mM dNTP mix, 10X RT buffer, 10 units/µl RNase inhibitor and 100 units/µl MMLV-RT) were then added to the reaction. Samples then underwent standard RT using the following conditions: 60 min at 42 ºC, 10 min at 92 ºC.

In all studies a negative control containing all components for reverse transcription except the sample template was prepared. A negative control containing all components except Moloney Murine Leukaemia Virus reverse transcriptase (MMLV-RT) was also prepared.

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2.5.2 Stem-loop reverse transcription

The TaqMan® microRNA reverse transcription kit and TaqMan® microRNA assays (Life Technologies, UK) were used for miRNA transcripts. The protocol used was the “TaqMan®_{small RNA protocol” [246].}

A RT master mix containing nuclease-free water, 10X reverse transcription buffer, 50 U/ l MMLV-RT, 20 U/ l RNase inhibitor and 100 mM dNTPs was prepared. The mastermix, 5X primer and extract was then combined into a single tube. Samples then underwent stem- loop RT on a Veriti thermocycler with the following cycling conditions: 16 °C, 30 min at 42 °C, 5 min at 85 °C and held at 4 °C. In all studies the same negative controls prepared as those in the standard reverse transcription protocol.

2.6 Quantitative PCR

As mentioned previously, there is currently no reliable method for quantifying RNA. An alternative method of quantification is through the comparison of gene expression levels via qPCR e.g. mRNA and miRNA. There are two different types of chemistries that can be used for qPCR analysis including Life Technologies TaqMan® chemistry and SYBR® Green chemistries. Many of the forensic research groups utilise these chemistry for BFID due to its high specificity and sensitivity and as such, has been selected for the development of this BFID test. Previous research by the University of Huddersfield research team explored SYBR Green chemistry for RNA-based BFID analysis. Findings from their

80 studies showed lack of specificity to mRNA targets and as such, TaqMan chemistry was explored in this work.

2.6.1 qPCR of stem-loop RT products

The “TaqMan® _{small RNA protocol” used in the cDNA synthesis section 2.5.2 was also}

used for the qPCR section. A single master mix containing 2X TaqMan® universal PCR master mix II no UNG (Life Technologies, UK) and nuclease free water was prepared in triplicate for each experiment in sterile 1.5 ml tubes (shown in Table 2). A 20X primer solution containing a TaqMan probe, a specific forward primer and universal reverse primer for miRNA (Ambion, UK) and RT product were then prepared in separate 0.2 ml PCR tube. A negative control containing all components of the PCR reaction apart from RT product was included in all studies.

Samples were then transferred to MicroAmp® optical 96-well reaction plate (Life Technologies, UK) and sealed with a MicroAmp® clear adhesive film and applicator (Life Technologies, UK). Quantitative PCR was performed on a 7500 fast real-time PCR system (Life Technologies, UK) using the following PCR cycling conditions: enzyme activation for 10 min at 95 °C and 40 cycles of denaturation for 15 s at 95 °C, annealing for 60 s at 60 °C and an extension during the temperature ramp of annealing and denaturation steps.

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QPCR MASTER MIX COMPONENTS 1 REACTION IN

TRIPLICATE ( l)

10 REACTIONS IN

TRIPLICATE ( l)

TAQMAN® UNIVERSAL PCR MASTER

MIX II (2 X) NO UNG

40.00 400.0

NUCLEASE-FREE WATER 30.68 306.8

TOTAL VOLUME _70.68 706.8

Table 2. Showing an example of a calculated qPCR master mix when using a total of 10 reactions.

2.6.2 qPCR of standard RT products

The “TaqMan® _{universal master mix II” protocol (Life Technologies, UK) was used to}

amplify RT product. A qPCR reaction mix containing 2X TaqMan® universal PCR master mix II no UNG, RT product or nuclease-free water and 20X TaqMan® primer assay was prepared in triplicate. A PCR negative control was also included in each study. Quantitative PCR reaction mixes were then transferred onto a 96-well plate and run on the fast 7500 qPCR machine with the following the same PCR cycling conditions as in the previous protocol.