Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization

Abstract

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.

Rapidly developing functional genomics in ecological model systems via 454 transcriptome sequencing

“…Size selection of cDNA is generally performed in an attempt to gain access to longer, full length transcripts, with some researchers cloning different size groups independently (e.g. Lévesque et al 2003).…”

Equine ANXA2 and MMP1 expression analyses in an experimental model of normal and pathological wound repair

“…The size of the full-length equine ANXA2 cDNA was estimated by virtual Northern blot analysis. Briefly, total RNA was isolated from a wound edge biopsy obtained 7 days following creation of a full-thickness wound on the lateral thoracic wall [4] and transformed into cDNA by the SMART cDNA synthesis method (BD Biosciences Clontech, Mississauga ON) as described [15]. The cDNA was separated by gel electrophoresis, transferred onto a nylon membrane and hybridized with an equine radioactive probe (ANXA2 = 266 bp: [GenBank: DN625870]) generated from a previous gene expression profiling experiment [4].…”

“…The cDNA was separated by gel electrophoresis, transferred onto a nylon membrane and hybridized with an equine radioactive probe (ANXA2 = 266 bp: [GenBank: DN625870]) generated from a previous gene expression profiling experiment [4]. On the basis of the hybridized size, a specific library was established via the pDrive plasmid cloning technique (Qiagen PCR cloning kit; Qiagen, Mississauga, ON) and screened by radioactive hybridization as described [15]. Positive hybridizing bacterial colonies were grown, their plasmid contents were isolated (QIA-prep, Qiagen) and the size of the cloned cDNA was analyzed via gel electrophoresis analysis following EcoR1 digestion.…”

Structure of the bovine VASAP-60/PRKCSH gene, functional analysis of the promoter, and gene expression analysis

“…Total RNA (1 μg) from granulosa cells (SF, DF, OF) or CL was reverse-transcribed using SMART PCR cDNA synthesis technology (BD Bio-Sciences Clontech, Mississauga, ON) in a total volume of 10 μl with the addition of 42 ng of T4 gene 32 protein (Roche Applied Science), oligo-dT30 primer (5′-AAGCAGTGGTAACAACGCAGAGTACT (30) (A/C/G/T) (A/ G/C)-3′), oligo IIA (5′-AAGCAGTGGTATCAACGCAGAG-TACGCGGG-3′) and PowerScript enzyme as described previously (Lévesque et al, 2003). The resulting cDNA pool was diluted to 50 μl in TE buffer (10 mM EDTA, 100 mM TrisCl, pH 8), and 1 μl of the diluted pool was used in a 100 μl PCR reaction for 18 cycles using Advantage 2 DNA polymerase and the PCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′; BD Bio-Sciences Clontech).…”

“…The cDNA products were separated on a 0.8% TBE-agarose gel (45 mM Tris-borate, 1 mM EDTA, 0.5 μg/ml ethidium bromide), transferred onto nylon membrane (Hybond-N + ; GE Healthcare Bio-Sciences Inc) by alkaline capillary transfer, and then cross-linked by UV treatment (Sambrook and Russel, 2001) to generate a virtual northern membrane. This membrane was hybridized as previously described (Lévesque et al, 2003) with probes labeled with [α-32 P]-dCTP corresponding to the full-length bovine VASAP-60 cDNA (2.1 kb) (Brûlé et al, 2000), stripped and hybridized with probes corresponding to the first 821 bp of the open reading frame of the human αGII (Treml et al, 2000), and bovine GAPD as described (Lévesque et al, 2003). Membranes were exposed to a phosphoscreen and the images were digitized (Storm 840; GE Healthcare Bio-Sciences Inc).…”