TROD HELP
rev 0.2
Table of Contents
1. Introduction to TROD
What is TROD?
What does it do?
2. Using TROD
User Input
Options
The Results Page
Interpreting Results
What do I order?
3. Credits
References
Copyright and License
How do I cite TROD?
4. FAQ
Chapter 1. Introduction to TROD
What is TROD?
Current Version: 1.1.0
TROD (T7 RNAi Oligo Designer) is a web application that facilitates the design of DNA oligonucleotides for the
synthesis of short interfering RNAs (siRNAs) with T7 RNA polymerase. We highly recommend reading the following
references that describe and review the technique in detail (go to the References section for more information): Donzé
and Picard (2002), Donzé et. al, (2004).
What does it do?
In brief: TROD takes a cDNA sequence as input and automatically generates the appropriate DNA oligos for ordering.
The output is organized so as to make it easy for the user to choose appropriate target sequences.
In detail: TROD (by default) looks for all occurrences of the sequence N2 GN 18C. The G and C nucleotides are required
for efficient synthesis of the RNA by T7 RNA polymerase, since it strongly favours a G at the start site. From these
sequences, the program generates both the sense and antisense strands of DNA oligos that will be used to produce the
siRNAs in vitro. By definition, the 'antisense' DNA oligo encodes the antisense siRNA strand, and vice versa.
The following (reverse complemented) T7 promoter is appended to the 3' ends of the DNA oligos: 5'TATAGTGAGTCGTATTA-3'.
After T7 transcription, the siRNA duplexes will look like the following:
sense siRNA:
5'-GNNNNNNNNNNNNNNNNNNUUU-3'
antisense siRNA: 3'-NNCNNNNNNNNNNNNNNNNNNG-5'
Important note: TROD, by default, appends an AA dinucleotide to the 5' end of the DNA 'sense' strand, since ATrichness is preferred at this site. The result (as shown above) is a UU dinucleotide overhang on the sense siRNA after T7
transcription. In addition, in order to destabilize the 5' end of the antisense siRNA (so as to direct it to the RISC
complex), a U replaces the complementary C, producing a GU pair.
Chapter 2. Using TROD
User Input
Users have the possibility of inputing a cDNA sequence in three different ways. Clicking on the submit button will launch
the program. Details and limitations for each input are described below:
GenBank Accession Code - All GenBank accession codes are supported apart from the ones starting with "NT_",
since NT contigs contain references to clones, rather than sequences. In the future, this will be remedied.
Sequence File - Any cDNA sequence in FASTA format is supported. Text files containing a single sequence are
also supported. In the future, many more file formats will be supported.
cDNA Sequence - A large text field is provided for pasting sequences directly.
Options
Several options have been made available. They are described below.
siRNA length - this refers to the length of the in vitro transcribed siRNA, which ranges from 21-24 nucleotides.
The default value is 22 nt, the length that we recommend.
Sort by - the user can choose to sort the output either by GC content (default) or by the location of the target
sequence in the cDNA.
The Results Page
Here is a screenshot of a sample Results Page, with explanations for each column below.
ID - each pair of oligos is given a unique identification number
DNA 'antisense' + T7 - this is the DNA oligo (with appended reverse-complemented-T7 promoter) that will be used to
generate the interfering (in other words, the 'antisense') siRNA strand
DNA 'sense' + T7 -this is the DNA oligo (with appended reverse-complemented-T7 promoter) that will be used to
generate the 'sense' siRNA strand
Location - the location in your cDNA sequence input of the target sequence (see "Sequence" column)
% GC - the calculated GC content of the target sequence (see "Sequence" column)
Sequence - the sequence in your cDNA that will be targeted by the siRNA duplex
Blast - provides a link to BLAST the targeted sequence (from the "Sequence" column). It is highly recommended to
BLAST your chosen sequences to ensure target specificity
- the DNA 'antisense' oligos that produce an AT-rich 3'-dinucleotide overhang on the antisense siRNA
Highlighted
strand as well as an AT-rich internal dinucleotide sequence adjacent to the 5' G are highlighted, since they
Rows
are the most desired
Interpreting Results
The results table has been organized to make it convenient for the user to choose ready-to-order oligos. The list of oligos
is sorted by increasing GC content (by default), while those that have an AT-rich 5'-dinucleotide on the DNA 'antisense'
oligo (as well as AT-richness adjacent to the 3' C) have been highlighted (in other words, the DNA oligos that produce
the following antisense siRNA: 5'-GW2 N17-20CW2 -3', where W = A or T). Although there are no well defined criteria
for choosing the "right" oligos, the following guidelines and recommendations should help:
choose oligos with GC contents of 30-50% (below or higher have been known to work but the probability is lower)
choose oligos with an AT-rich 5'-dinucleotide on the 'antisense' DNA strand as well as AT-richness near the 3' C
(these have been highlighted!)
try to avoid long stretches (>3) of any single nucleotide (but especially G)
remember to always BLAST the sequence to ascertain target specificity!
What do I order?
The in vitro transcription step requires 3 oligos: [1] DNA 'antisense' oligo, [2] DNA 'sense' oligo, [3] T7 promoter.
Ordering is as simple as copying and pasting the sequences directly from the Results Page. The sequence of the required
T7 promoter oligo is shown below:
T7 Oligo (5'-3'): TAATACGACTCACTATAG
Chapter 3. Credits
References
This article is the original description of the technique:
Donzé, O. and Picard, D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA
polymerase. Nucleic Acids Research, 30 (10): e46 [Pubmed Link]
This article reviews the technique, and includes very detailed protocols:
Donzé, O., Dudek, P., and Picard, D. (2004) siRNA production by in vitro transcription. In: Gene Silencing by RNA
Interference: Technology and Application, M. Sohail, ed. (CRC Press LLC), in press.
Copyright and License
The TROD program is copyright © 2003 Peter Dudek
The Documentation is copyright © 2003 Peter Dudek, Didier Picard
This program is licensed under the terms of the GNU General Public License
How do I cite TROD?
Information will be available soon.
Chapter 4. FAQ
1. Why are there so few (or no) highlighted oligos in the output?
There are a few reasons why this could occur. The most common reason is that your sequence is too GC-rich. Since
the highlighting "algorithm" favours AT-richness in certain regions of the oligo, the highlighting is biased towards
sequences that have more A/T's. However, all is not lost. One thing that may help increase the number of
highlighted oligos is changing the length of the siRNA at the options section of the program page. Another thing
one should keep in mind, however, is that just because it's not highlighted, doesn't mean it won't work! The oligos
don't necessarily need to meet all of the guidelines outlined in the "Interpreting Results" section. Many
"unhighlighted" oligos could still produce functional siRNAs.
2. Why don't the highlighted oligos print in colour?
This is due to your browser settings. By default, most browsers set the 'printing background colours' option to off.
You need to enable the printing of background colours to see the highlighted oligos in prints.
3. Can you please add feature/option X to the program?
If there's sufficient demand (or the change is in the best interests of the user), we may add it.
4. I found a bug - what should I do?
Please let us know! Email the relevant information (including how to reproduce it) to the author of the program.