|
|
| Deborah's "makeover" of draft application note text can be found below. A copy of the completed note is available upon request.
|
Client: GE Healthcare Biosciences (formerly Amersham Biosciences), Little Chalfont, UK.
Need: GE Healthcare Biosciences is a leading provider of medical diagnostics, drug discovery, and protein separation systems. Its "Application Notes" provide experimental support for the company's marketing claims, usually by detailing an experimental scenario highlighting a key product feature.
Solution: Deborah J. Ausman edits about four pieces per month for GE Healthcare. A "before/after" example is shown below. Deborah does more than simply proofread the documents--she often reorganizes them to highlight key claims; rewrites text to tout the benefits of the experimental results; and improves the presentation to make the note easier to follow. In this note, Deborah suggested adding some context to make clear the value of the new set of control probes through a footnote to a Nature correspondence that she found in the course of background research. She also removed the discussion of the signal ratio to the results/discussion part of the note in order to focus the introduction.
Copyright © 2004 by GE Healthcare. All rights reserved. Reprinted here by permission.
CodeLink 3'/5' Degradation Controls for Assessing Sample Quality
|
BEFORE
|
AFTER
|
|
This note describes the selection and application of a new set of CodeLink control probes for assessing RNA
sample quality. In addition to the conventional probe sets designed to be close in proximity to the 3’ end
of a transcript, additional probe sets in the 5’ region of the transcript have also been selected for certain
housekeeping genes. Signal intensity ratio of the 3' probe set over the 5' probe set is often referred to
as the 3'/5' ratio. This ratio gives an indication of the integrity of your starting RNA, efficiency of
first strand cDNA synthesis, and/or in vitro transcription of cRNA. Since the signal of each probe set, for
a given housekeeping gene, reflects the sequence of the probes and their hybridization properties a 1:1
molar ratio of the 3’ to 5’ transcript regions will not necessarily give a signal ratio of 1. There is no
single threshold cutoff to assess sample quality for all of the diverse organisms and tissues. This is due
to the presence of different isoforms of these house-keeping genes and their different expression patterns
in various tissues and organisms. Although we suggest a threshold ratio of less than 3 for the most common
tissues, such as mammalian liver and brain, this may not be applicable to all situations. It may be more
appropriate to document the 3'/5' ratios within a particular study and flag the results that deviate,
therefore representing an unusual sample that deserves further investigation.
|
This note describes the selection and application of a new set of control probes for the CodeLink™
Expression Bioarray System. Gene expression measurements assume that the analyzed RNA sample closely
represents the actual transcript amount present in vitro. RNA degradation, therefore, must be
identified in order to eliminate potential bias in transcript quantification (1).
A common measure of RNA degradation is a signal intensity ratio often referred to as the 3'/5' ratio. In
the case of the CodeLink degradation controls, conventional probes designed to be proximal to the 3' end of
the transcript were assessed and compared to a set of additional probes in the 5' region of certain
housekeeping genes. The 3'/5' ratios obtained for the control set offer a measure for identifying RNA
quality issues, including those caused by poor RNA starting material, inefficient first-strand cDNA
synthesis, or problems with in vitro cRNA transcription.
Note: The "before" text discussing the vagaries and application of the 3'/5' ratio was removed to the results/discussion section of the note.
|
| |
