Writing Technique: Break up a Single Paragraph into Two
Source: Introduction from Chomczynski et al. Anal. Biochem. 162, 1987, 156-9
This Before and After post demonstrates how breaking up a single block of text into two paragraphs can provide helpful visual landmarks for the reader. Notice how the first sentence of the second paragraph tells you where look for the background information versus the authors’ contributions. Word choices are made differently on either side across this important sentence.
BEFORE
Guanidinium thiocyanate and chloride are among the most effective protein denaturants (1,2). As a strong inhibitor of ribonucleases, guanidinium chloride was first introduced as a deproteinization agent for isolation of RNA by Cox (3). Since then guanidinium extraction has become the method of choice for RNA purification, replacing phenol extraction. Guanidinium methods have been used successfully by Chirgwin et al. (4) to isolate undegraded RNA from ribonuclease-rich tissues like pancreas. Chirgwin’s protocol for ultracentrifugation of a guanidinium thiocyanate lysate through CsCl cushion has become one of the most frequently used isolation of undegraded RNA. In the present report, a new rapid procedure combining guanidinium thiocyanate and phenol-chloroform extraction is described. A combination of guanidinium and hot phenol for RNA isolation has been reported by Fermisco et al. (5). The method we describe differs in that it converts the guanidinium-hot phenol method to a single step extraction which allows isolation of RNA in 4 h and provides both high yield and purity of undegraded RNA preparations. By eliminating the ultracentrifugation step the guanidinium-CsCl method this procedure allows the simultaneous processing of a large number of samples. In addition, this new procedure permits recovery of total RNA from small quantities of tissues or cells making it suitable for gene expression studies for which only a limited quantity of biological material is available.
AFTER
Current RNA extraction methods that use guanidinium thiocyanate and chloride as deproteinization agents are outcompeting older methods such as phenol extraction (1-3). Recent breakthroughs have used the ultracentrifugation of a guanidinium thiocyanate lysate through CsCl cushion to extract undegraded RNA from ribonuclease-rich tissues, expanding the capabilities of guanidinium methods (4). However, the need for a ultracentrifugation step still hinders simultaneous processing of samples in large number, greatly reducing the RNA yield.
Here, we describe a new procedure that combines guanidinium and phenol extraction methods. Using guanidinium thiocyanate and phenol-chloroform, our approach converts a previously established guanidinium-hot phenol method (5) into a single-step procedure, extracting high-yield, high-purity, undegraded RNA in four hours. We show that, by eliminating the ultracentrifugation step, our approach can process a large number of samples simultaneously. We also show that the efficiency of our procedure can achieve complete RNA recovery from small quantities of tissues or cells, making gene expression studies of samples with limited biological materials possible.