The production of a high quality library containing a large number of full-length cDNA clones requires that the tissue and RNA preparation be performed in a certain way. These methods will help to guarantee that the starting material and ultimately the cDNA library are not compromised.
To minimize RNA degradation, all tissues and cells need to be prepared as quickly as possible and flash frozen in liquid nitrogen. These samples should be stored at -80°C and shipped packed in a large quantity of dry ice.
If RNA is to be isolated, the frozen tissues should be ground to a fine powder using liquid nitrogen and a mortar and a pestle. To isolate RNA from cells, the cells should be pelleted and the pellet washed with PBS. Total RNA should be quickly isolated by sample homogenization or lysis using a reagent, such as TRIzol or equivalent. Total RNA can be further processed to mRNA using most commercial kits. Total RNA or mRNA should be dissolved in nuclease-free water (no DEPC) at a concentration of ≥1 µg/µl. The mRNA to be used in the construction of microquantity libraries may be sent at 100 ng/µl. For nanoquantity libraries, the total RNA may be sent at 25 ng/µl and the mRNA at 1 ng/µl. The A260/A280 ratio must be at least 1.8. RNA should be stored at -80°C and shipped packed in a large quantity of dry ice. Please include a gel photo of the total RNA in the shipment. An indicator of total RNA quality is the 28S rRNA band is nearly twice the intensity of the 18S rRNA band.
Depending on the type of cDNA library that is desired, different amounts of tissue, cells or RNA will be needed (see below).
cDNA Library Requirements
| Type of cDNA Library | Amount of Tissue | Number of Cells | Amount of Total RNA | Amount of mRNA |
|---|---|---|---|---|
| Standard | 1 g | 1×108 | 1 mg | 5 µg |
| Large Insert | 2 g | 2×108 | 2 mg | 10 µg |
| Microquantity | 100 mg | 1×107 | 50 µg | 500 ng |
| Nanoquantity | 250 µg | 25,000 | 250 ng | 5 ng |
Type of cDNA Library, Amount of Tissue, Number of Cells, Amount of Total RNA, Amount of mRNA*For your convenience, our qualified staff can isolate mRNA from your total RNA, tissue or cell samples.