Quantitative real-time PCR and RT-PCR

Standard targets for the amplification reactions were obtained from plasmid pHR0, that contains an insert corresponding to the complete internal region of B19V genome (nt. 346–5245) [19]. From pHR0 plasmid, in vitro amplified DNA or in vitro transcribed RNA corresponding to the viral insert were obtained, purified, quantified and serially diluted to obtain calibration standards. Primers and primer combinations used in the qPCR and qRT-PCR assays are indicated in Table 1 and located with respect to the functional map of B19V genome in Fig 1 [3,20,21]. All oligonucleotides were obtained from MWG Biotech.

Top, ORF distribution within B19V internal coding region and related coding sequences. Center, functional map of B19V genome and distribution of regulatory signals: grey boxes, inverted terminal regions; P₆, promoter region; pAp1, pAp2, pAd: proximal and distal cleavage-polyadenylation sites; D1, A1-1/2, D2, A2-1/2, donor and alternative acceptor sites for introns 1 and 2, respectively. Below, a simplified map of B19V transcripts. Bottom, position of primers used in the PCR array (Table 1).

Sequence of primers is derived from reference genome sequence {"type":"entrez-nucleotide","attrs":{"text":"NC_000883","term_id":"356457872","term_text":"NC_000883"}}NC_000883, as complementary either to a contiguous sequence or to a non-contiguous sequence. In primers marked with *, bases on the 5’ end are complementary to bases on the external side of a splice site, whereas bases on the 3’ end shown in italics are complementary to bases on the internal side of a splice site. RNA target is defined with respect to the functional map of B19 virus genome (Fig 1).

Real-time PCR and RT-PCR were carried out by using the RotorGene 3000 system (Corbett Research) and SybrGreen detection of amplification products. Amplification reactions were performed by using QuantiTect PCR SybrGreen PCR Kit (Qiagen) or QuantiTect SybrGreen RT-PCR Kit (Qiagen), including 10 pmol of each specific primer pair. For PCR, thermal profile consisted in 15 min at 95°C, then 40 cycles of 15 sec at 95°C, 30 sec at 55°C, and 30 sec at 70°C coupled with signal acquisition. For RT-PCR, two parallel reactions were performed for each sample, either including (RT+) or omitting (RT-) the reverse transcriptase from the reaction mix, and performing an initial step consisting in 30 min at 55°C, before the amplification reaction with a standard thermal profile. A final melting curve was performed, with thermal profile ramping from 50°C to 95°C at a 12°C/min rate, coupled with continuous signal acquisition.

Fluorescence emission recorded in the FAM/Sybr channel of the instrument was analyzed by using the functions available in the RotorGene 6.0 software. All reported experiments were carried out in duplicate series, and melting curve and quantitative analysis performed. Melting curve analysis was used for the determination of the specificity of the amplification products by defining, for each reaction, the melting profile and the Tm of the products.

For control and normalization with respect to the number of cells, a target sequence in the region of genomic DNA coding for 18S rRNA (rDNA) was selected and amplified by using the primer pair 18Sfor/18Srev. For quantitative evaluation of viral targets, the pair R2210-R2355, located in the central exon of B19V genome, was used for amplification of both viral DNA and total viral RNA, and absolute quantitation of viral DNA and total viral RNA was obtained by calibration to the standard DNA and RNA targets. For RNA targets, values for both RT+ and RT- reactions were obtained, and interference by amplification of residual DNA was evaluated by subtracting the residual RT- from the RT+ values. For the determination of the relative abundance of the different species within the whole set of viral transcripts, an array of primer pair combinations was used in group sets as indicated in Table 1. Relative quantitation of the different subsets of viral transcripts was obtained by efficiency-corrected comparative quantitation using LinRegPCR software [22], and subsequent normalization within each combination set and to the amount of total viral RNA. Data analysis was carried out by using the program GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California, USA).