[Frontiers in Bioscience 2, c1-5, February 15, 1997]

	[Frontiers in Bioscience 1, c4-15, November 1, 1996] Reprints PubMed CAVEAT LECTOR
	EXPERIMENTAL PROCEDURE FOR THE DETECTION OF A RARE HUMAN mRNA WITH THE DIG SYSTEM Barbara Rueger, Julia Thalhammer, Irmgard Obermaier, and Stefanie Gruenewald-Janho Boehringer Mannheim Customer Service Lab, International Support, Mannheim, Germany 3. RESULTS AND DISCUSSION Using the procedure described here, we were able to detect the highly abundant mRNA for ß-actin in as little as 5 ng of total RNA in a 15 min exposure. This procedure also allowed us to detect the low abundant mRNA for the CTF1 transcription factor in 50 ng total RNA in just 15 min (Figure 1). We have only used standard procedures as described in our DIG System User's Guide for Filter Hybridization and package inserts. We take several main precautions when working with RNA: keeping everything as sterile as possible, using non-powdered gloves, and handling the positively charged membrane with extreme care. We strictly follow the package insert instructions. With the technique summarized in Table 2, we achieve an extraordinarily high sensitivity compared to radioactive hybridizations. In contrast to the 3-4-day exposures required to detect RNAs in at least 2-5 µg poly(A)⁺ RNA with a radioactive probe, the DIG System produces specific signals in 50 ng after just a 15 min exposure (Figure 1). Figure 1. Northern blots detected with DIG-labeled RNA blots. The procedure described in the text and summarized in Table 2 was performed with CSPD chemiluminescent substrate, and the blot was exposed to X-ray film for 15 min A. Hybridization with ß-actin probe B. Hybridization with CTF1 probe Target: Lane 1: 100 ng DIG-labeled RNA Molecular Weight Marker Ill Lane 2: 100 ng human skeletal muscle total RNA Lane 3: 50 ng human skeletal muscle total RNA Lane 4: 5 ng human skeletal muscle total RNA Lane 5: 1 ng human skeletal muscle total RNA

[Frontiers in Bioscience 1, c4-15, November 1, 1996]
Reprints
PubMed
CAVEAT LECTOR

EXPERIMENTAL PROCEDURE FOR THE DETECTION OF A RARE HUMAN mRNA WITH THE DIG SYSTEM

Barbara Rueger, Julia Thalhammer, Irmgard Obermaier, and Stefanie Gruenewald-Janho

Boehringer Mannheim Customer Service Lab, International Support, Mannheim, Germany

3. RESULTS AND DISCUSSION

Using the procedure described here, we were able to detect the highly abundant mRNA for ß-actin in as little as 5 ng of total RNA in a 15 min exposure.

This procedure also allowed us to detect the low abundant mRNA for the CTF1 transcription factor in 50 ng total RNA in just 15 min (Figure 1). We have only used standard procedures as described in our DIG System User's Guide for Filter Hybridization and package inserts. We take several main precautions when working with RNA: keeping everything as sterile as possible, using non-powdered gloves, and handling the positively charged membrane with extreme care. We strictly follow the package insert instructions.

With the technique summarized in Table 2, we achieve an extraordinarily high sensitivity compared to radioactive hybridizations. In contrast to the 3-4-day exposures required to detect RNAs in at least 2-5 µg poly(A)⁺ RNA with a radioactive probe, the DIG System produces specific signals in 50 ng after just a 15 min exposure (Figure 1).

Figure 1. Northern blots detected with DIG-labeled RNA blots. The procedure described in the text and summarized in Table 2 was performed with CSPD chemiluminescent substrate, and the blot was exposed to X-ray film for 15 min

A. Hybridization with ß-actin probe

B. Hybridization with CTF1 probe

Target:

Lane 1: 100 ng DIG-labeled RNA Molecular Weight Marker Ill

Lane 2: 100 ng human skeletal muscle total RNA

Lane 3: 50 ng human skeletal muscle total RNA

Lane 4: 5 ng human skeletal muscle total RNA

Lane 5: 1 ng human skeletal muscle total RNA