Molecular Research Center Tech Bulletin - Microcarrier Gel and Absorbance Readings

Hybridization Molecular Research Center Technical Bulletin No. 3

MICROCARRIER GEL CONTRIBUTION TO ABSORBANCE READINGS
Karol Mackey, Molecular Research Center, Inc.

Precipitation often becomes a limiting factor when isolating small quantities of nucleic acids (approximately 20 µg or less). For this reason, a common practice involves the addition of a carrier to facilitate precipitation of RNA and/or DNA. Transfer RNA and glycogen are two commonly used carriers. Molecular Research Center has developed two polysaccharide-based carriers for nucleic acid precipitation, Microcarrier ^TM Gel (Cat.No. MG 122) and Microcarrier^TM Gel-TR (Cat.No.MR 123). Microcarrier Gel can be used in a variety of settings to facilitate RNA or DNA precipitation. Microcarrier Gel-TR is specially designed for use with TRI Reagent products. Microcarrier Gel-TR is added at the beginning of an isolation procedure when cells or tissues are lysed or homogenized in the reagent. This carrier facilitates the isolation and precipitation of both RNA and DNA during the course of the procedure.
Although carriers promote an increased nucleic acid yield, they present an interfering factor by contributing to the optical density readings that are commonly used to quantify RNA and DNA yields. In a like manner, Microcarrier Gel-TR contributes to the 260 and 280 absorbance readings for RNA isolated using TRI Reagent. (DNA absorbance readings are not affected.) For this reason, we suggest the following procedure to normalize optical density readings of RNA isolated in the presence of Microcarrier Gel-TR.

We recommend using 5 - 8 µl of carrier with 0.8 - 1.0 ml of TRI Reagent.
Prepare a blank that contains only reagent and carrier without any biological sample. Process this blank according to protocol (identical to experimental samples) and solubilize the resulting pellet in the same volume used for experimental RNA pellets.
Measure the optical density at 260 and 280 nm for all samples and the blank.
The absorbance at 260 and 280 nm for the blank can then be subtracted from the values obtained for each respective sample. In this manner, normalization of the optical density readings allows a more accurate determination of the amount of RNA in each sample.