Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.
Part 1: cDNA synthesis from RNA
Part 2: Double-stranded cDNA clean-up
Part 3: in vitro Transcription (IVT)
Part 4: aRNA clean-up after IVT
Table 1: cDNA 1st Strand Synthesis Master Mix
Reagent | Amount for 1 reaction |
10X First Strand Buffer | 2 µl |
dNTP Mix | 4 µl |
Ribonuclease Inhibitor | 1 µl |
Array Script | 1 µl |
Table 2: cDNA 2nd Strand Synthesis Master Mix
Reagent | Amount for 1 reaction |
Nuclease free water | 63 µl |
10X Second Strand Buffer | 10 µl |
dNTP Mix | 4 µl |
DNA Polymerase | 2 µl |
RNase H | 1 µl |
Table 3: IVT Master Mix
Reagent | Amount for 1 reaction |
aaUTP solution (75 mM) | 2 µl |
ATP, CTP, GTP mix (25 mM) | 12 µl |
T7 UTP solution (75 mM) | 2 µl |
T7 10X Reaction Buffer | 4 µl |
T7 Enzyme Mix | 4 µl |
Table 4: aRNA Binding Mix
Reagent | Amount for 1 reaction |
RNA Binding Beads* | 10 µl |
Bead Resuspension Solution* | 4 µl |
100% isopropanol** | 6 µl |
aRNA Binding Buffer Concentrate | 50 µl |
* Mix the RNA binding beads with the bead resuspension solution first.
** Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.
Critical Steps
A second round of amplification is not advised as biases in array data have been observed. Mixing carefully at each enzymatic step (1st and 2nd strand cDNA synthesis, IVT) is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature. A PCR cycler with adjustable heated lid is preferred – even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridization can significantly affect yield of amplified product.
Application/Significance
The labeled RNA resulting from this protocol can be hybridized to human, viral, or custom microarrays to assess gene expression responses to infected cells in culture. Microarray platforms vary, so follow manufacturer instructions for preparation of hybridization mixture from labeled probe.
Using a custom designed poxvirus array1, we were able to classify genes into the general categories of “early” or “late” based on timing of hybridization signal and whether or not viral DNA replication was required for transcript detection. We observed the expected functional categories of genes in each temporal class (i.e., expected early, intermediate and late genes) variation as to the exact timing of transcription.
The methods utilized in this work are able to predict virus genes transcribed early or late in the replication cycle, but have more difficulty distinguishing early-only versus genes with an early and late promoter since transcripts with a dual early/late promoter may persist and be detected at late times. In addition, run-through transcription of late viral genes may affect signal at a given probe/spot on the array, as the RNA hybridizing to the array may have come from the designated ORF or an upstream ORF. Tiling arrays have attempted to resolve this issue, however challenges remain in detecting run through transcription using hybridization based approaches2,3,4.
Host transcriptional patterns can also be assessed using these methods. However, vaccinia encodes a variety of mechanisms to inhibit host responses, and host transcriptional responses may be diminished compared to other stimuli5,6,7,8. Since the expression of many genes involved in host defense is altered after infection, the contribution of viral genes that counteract host immune responses should therefore be taken into consideration.
Utilizing these methods, a map of the transcriptional timing of all viral genes can be identified and used to interrogate functions of unknown viral genes. In addition, these methods can be utilized to dissect the intricate dialogue between virus and host. These methods are broadly applicable to other host-pathogen infection systems. If the pathogen of interest does not have polyadenylated mRNAs, alternative methods can be used to directly label the total RNA, without linear amplification. By analyzing both host and virus gene expression during synchronous infection, these methods allow us to gain insight into virus interaction with the host cellular environment as well as host counter-defenses against virus infection.
The authors have nothing to disclose.
Whitehead Institute Fellows Funds
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Amino Allyl MessageAmp II aRNA amplification kit | Reagent | Applied Biosystems | AM1753 | For 20 reactions |
Amino Allyl MessageAmp II aRNA amplification kit | Reagent | Applied Biosystems | AM1821 | For 100 reactions, in a 96-well format |
NanoDrop ND-1000 UV-VIS spectrophotometer | Other | NanoDrop | ND-1000 | Or equivalent spectrophotometer |