Normalized cDNA Libraries

Improve the Quality of Yeast Two-hybrid Screens

You may also view our non-normalized cDNA libraries

A few abundant transcripts dominate RNA populations

High abundance transcripts, such as housekeeping genes and components of the transcriptional and translational machinery make up 80-90% of all mRNAs. These transcripts are expressed at very high levels in a cell and represent the most frequent class of false positive interactions in yeast two-hybrid screens.

Low abundance transcripts encode many of the key signalling proteins of a cell. They are transcribed at very low levels, making up less than 1% of the total mRNA population. Yet, they encode key components and are often the main targets of protein interaction hunts. Constructing cDNA libaries which represent

low abundance transcripts is the central challenge of yeast two-hybrid screening. Results show that significantly fewer clones have to be screened with a normalized library to identify relevant, high stringency interactions. Simultaneously, many low stringency false positive interactions are removed (comparing the number of primary interactions), resulting in an overall "cleaner" screen.

Unique improvements

These high quality cDNA's are constructed using a brand new technology. Dualsystems utilizes the specific features of MMLV-based reverse transcriptase to create full length enriched double stranded cDNA from total or polyA+ RNA samples. Differences in length of the 5’ adaptor primer ensure transcription of all three reading frames.

A double stranded cDNA sample is normalized based on differential re-association kinetics after denaturation since abundant transcripts re-associate faster than rare ones. Transcripts are denatured and then reannealed under carefully controlled conditions. The reannealing process is stopped after a short time, when the majority of abundant transcripts have reformed into double stranded cDNA, whereas rare transcripts are still single stranded. Subsequently, double stranded cDNA is removed by digestion and the remaining transcripts are denatured again and annealed to double stranded cDNA. The resulting cDNA population is normalized, i.e. the majority of abundant transcripts have been removed. Finally, the normalized cDNA sample is size selected and ligated into the appropriate yeast two-hybrid vector. Normalization of cDNA is confirmed by qPCR using two high abundant marker genes (Figure 1). Normalized cDNAs are amplified by representational PCR and cloned directionally into the library vector of choice.

Results you can measure

Figure 1: qPCR of normalized and non-normalized human spleen cDNA. For Actin, a decrease of 75.8% was reached. For hGAPDH, the decrease was 60.6%. The lower amount of these two high abundant cDNAs increases the quality of the cDNA library.

Packaged for success

Libraries are supplied as 200 µg or 60 µg purified plasmid DNA that is ready for screening. 200 µg is sufficient for seven library scale screens and 60 µg is sufficient for two library scale screens. cDNAs are cloned into pGAD-HA or pGAD-DS (N-terminal GAL4-fusion) for DUALhybrid libaries or pPR3-N (N-terminal NubG-fusion) or pPR3-C (C-terminal NubG-fusion) for DUALmembrane & DUALhunter libaries.

Yeast two-hybrid (DUALhybrid) Normalized cDNA libraries

ITEM #	SIZE	DESCRIPTION	PRICE	BUY	DATA SHEET
P02401	200 µg	Normalized mouse brain adult	$1,720
P12401	60 µg	Normalized mouse brain adult	$860
P02402	200 µg	Normalized human fetal brain	$1,720
P12402	60 µg	Normalized human fetal brain	$860
P02403	200 µg	Normalized Universal Arabidopsis Thaliana	$1,720
P12403	60 µg	Normalized Universal Arabidopsis Thaliana	$860

Yeast two-hybrid Normalized (DUALmembrane & DUALhunter) cDNA libraries

ITEM #	SIZE	DESCRIPTION	PRICE	BUY	DATA SHEET
P02301	200 µg	Normalized mouse whole embryo 17 days / x-NubG	$2,900
P12301	60 µg	Normalized mouse whole embryo 17 days / x-NubG	$1,290
P02302	200 µg	Normalized human spleen / NubG-x	$2,900
P12302	60 µg	Normalized human spleen / NubG-x	$1,290
P02303	200 µg	Normalized mouse brain / NubG-x	$2,900
P12303	60 µg	Normalized mouse brain / NubG-x	$1,290
P02304	200 µg	Normalized human fetal brain / NubG-x	$2,900
P12304	60 µg	Normalized human fetal brain / NubG-x	$1,290
P02305	200 µg	Normalized universal arabidopsis / NubG-x	$2,900
P12305	60 µg	Normalized universal arabidopsis / NubG-x	$1,290
P02306	200 µg	Normalized mouse brain / x-NubG	$2,900
P12306	60 µg	Normalized mouse brain / NubG	$1,290