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LIBRARY CONSTRUCTION

library construction

cDNA LIBRARY CONSTRUCTION

cDNA libraries identify all expressed genes in a tissue. Gene expression, also called protein expression or often simply expression is the process by which a gene's DNA sequence is converted into the structures and functions of a cell. Gene expression is a multi-step process that begins with DNA, which genes are made of, into messenger RNA. It is then followed by post transcriptional modification and translation into a gene product, followed by folding, post-translational modification and targeting. The amount of protein that a cell expresses depends on the tissue, the developmental stage of the organism and the metabolic or physiologic state of the cell. Thus, each library will represent a “snapshot” in time of the activity in the particular cells. Each library starts with 1.0 ug of high quality total RNA. Steps involved in the creation of the libraries include double-strand cDNA preparation and amplification, purification to remove primer excess, etc., hybridization and fractionation of the double-strand cDNA, and normalization (if requested) followed by two amplifications.

Sanger sequencing. If a small number of clones are to be sequenced, we clone the library into a vector. The clones are picked, grown and sequenced. The number of clones to be sequenced is determined by the customer, but sequencing must be done in batches of 96.

454 sequencing. Preparation includes amplification of sufficient quantities and purification of the library. A titration run is made, then 1-4 production runs may be specified.

  • Normalized library (LIB-NORM-SPE):  Each cell is known to express from about 10,000 to 50,000 genes, and transcript abundance varies from 200,000 copies to 1 or fewer copies per cell. As a rule, each cell expresses 10-20 highly abundant genes (several thousands of mRNA copies per cell), several hundreds of genes of medium abundance (several hundreds of mRNA copies per cell), and several thousands of rare genes (from one to several dozens of mRNA copies per cell).  Hence, direct random sequencing of clones from standard cDNA libraries is inefficient for discovering rare transcripts, because cDNAs of medium and high abundance are sequenced repeatedly instead. Normalization performed before cDNA library sequencing decreases the prevalence of clones representing abundant transcripts. Thus, normalization increases the efficiency of random sequencing dramatically, and is essential for rare gene discovery.
  • Non-normalized library (LIB-NN-SPE): In the non-normalized library, high and medium abundance genes have a greater chance of being sequenced than rare transcripts.  This approach may be useful for initial gene discovery using pooled samples of several different tissues, when little is known about an organism.  Both libraries (normalized and non-normalized) can be provided simultaneously for an additional fee.

Upon completion of the library, the customer will receive:

  • An Excel listing of all genes sequenced. All gene sequences belong to the customer. If an academic discount has been provided, EcoArray reserves the right to use these sequences for commercial use.
  • Glycerol stocks of the library.

Pricing: Please contact us for academic or commercial price.

SUPPRESSIVE SUBTRACTIVE HYBRIDIZATION (SSH) LIBRARY CONSTRUCTION

Suppression subtractive hybridization (SSH) is one of the most powerful and popular PCR-based methods for isolating differentially expressed transcripts. Using a small quantity of either total RNA or poly A+ mRNA (2.5 ug) from each of two populations, the SSH procedure simultaneously subtracts and partially normalizes the abundance of target cDNAs in the subtracted population. Two subtraction libraries, forward (“experimental”) and reverse (“control”), are generated by each subtraction protocol. The clones are ligated into a vector, plated, picked, and sequenced. The customer decides how many clones will be sequenced (in batches of 96), although we generally recommend sequencing 96 forward and 96 reverse clones (n=192). These genes are then available for analysis and further investigation by means of follow-on projects (e.g., creating a custom nylon membrane array or running real-time PCR).

Upon completion of the subtraction, the customer will receive:

  • An Excel listing of all genes sequenced. All gene sequences belong to the customer. If an academic discount has been provided, EcoArray reserves the right to use these sequences for commercial use.
  • Glycerol stocks of the forward and reverse libraries. If requested, glycerol stocks of the unsubtracted forward and reverse libraries.

With our flexible pricing, EcoArray can begin the suppression subtractive hybridization the procedure with tissue, blood, total RNA, or poly A+ mRNA that you provide. Pricing is based on how much of the preparation is done by the customer, how much starting material is provided, and how many clones are sequenced. We provide two options for suppressive subtraction hybridization:

  1. Standard subtraction: In this less expensive option, the customer provides (for each population to be subtracted)
    • Enough tissue or blood to obtain 500 ug of total RNA, or
    • 500 ug of high quality total RNA total, or
    • 2.5 ug of high quality poly A+ mRNA
  2. Limited starting material: When the tissue is at a premium and it is not possible to obtain any of the quantities listed for the standard subtraction, we can start with 1 ug of high quality total RNA.This process costs more as it takes several additional days to remove the excess ribosomal RNA and amplify the cDNA corresponding to the poly A+ fraction.

A typical standard subtraction experiment is described below:

Typical Subtraction Experiment: We performed a subtraction of FHM male livers, using tissue from 10 control fish and 10 that had been exposed to E2. We dissected out the livers, extracted total RNA from each liver separately, combined a 50 ug aliquot of total RNA from each liver and then converted it to poly A+ mRNA. The subtraction was run using 2.5 ug mRNA; 192 genes from forward and reverse (96 from each direction) were sequenced.

  • Forward subtraction: E2 exposed cDNA was the tester.
  • Results: Upregulated genes included vitellogenin and toxin-1.
  • Reverse subtraction: Control cDNA was the tester.
  • Results: Upregulated genes included proteasome subunit beta 7 and vesicular inhibitory amino acid transporter.

Please contact us via This e-mail address is being protected from spambots. You need JavaScript enabled to view it or this form for academic or commercial pricing.

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