Real time RT-PCR using SDS and RQ manager software and delta delta Ct method (9-29-2010) Yujiro Kida
(Equipments) ABI 7900 HT, MicroAmp® Optical 96-Well Reaction Plate (N8010560), MicroAmp® 96- & 384-Well Optical Adhesive Film (4311971), MicroAmp® Optical Film Compression Pad (4312639)
(Reaction mix) For example, when you examine 10 samples, prepare reaction mix for 11 samples. Prepare (1) primer mix including 5’ primer, 3’ primer and 2xSYBR green mix and (2) cDNA mix including cDNA and dH2O. Apply your each “primer mix” along the left (right) side wall of each well. Next, apply your cDNA mix along the right (left) side wall of each well. Seal 96 well plate with adhesive film tightly and centrifuge your 96-well plate to spin down all samples to the bottom of wells.
(Setting up 96-well plate in ABI 7900 HT)
(1) Open SDS software.
(2) Select [File] and [New]. The window of [New Document will appear.] Then select [delta delta Ct (RQ)], [96 Well Clear Plate] and [Blank Template]. Click [OK].
(3) Click [Add Detector], which you can find a middle lower part of the new window. The window of [Detector Manager] will appear. In this new window, select “gene of your interest” and click [Copy to Plate Document]. After completion of this step, click [Done]. If you need to create a new one, do the following steps. (A) Click [New] in the window of [Detector Manager]. The new window of [Add Detector] will appear. (B) Input the name of gene, select “SYBR” at the space of “Reporter”, “Non Fluorescent” at the space of “Quencher”. (C) Click [OK]. Then you can find your creating new name in the window of [Detector Manager].
(4) If you quantify an mRNA expression level of “gene A” using well A1-well A12, select well A1-A12. Then check [Use] of “gene A”. Repeat this step for every gene. Select “Endogenous Control” for your internal control gene such as GAPDH, HPRT1, 18S, etc. Otherwise task of all genes will be “target”.
(5) If you use “cDNA-1” in well A-1, A-2, B-1, and B-2 (duplication), select these wells. Input your sample name at the space of [Sample Name] (Default sample name is *Mixed*). Repeat this step for your all wells, which will make your later analysis easier. Passive Reference must be “ROX” and unused wells should be omitted by checking [Omit Well(s)].
(6) Click [Instrument] tab and set your PCR program as you want. Input “25” at the space of “Sample Volume (mL)”, if your sample volume is 25 mL per well. Save your assay.
(7) Click [Real-Time] tab and click [Connect to Instrument]. Then “Instrumental Status” will change to “Connected” Click [Open/Close]. Then the tray will come out. Place a compression pad on 96-well plate (Otherwise your samples will evaporate and results will be inaccurate). Set your 96 well-plate in the correct direction (well A1 must be left upper corner). Click [Start Run]. qPCR assay will automatically start.
(Analysis of your data)
(1) When your assay is done, click [OK] and save your data again.
(2) Open SDS RQ Manager. Select [File], [New Study] and [From SDS File]. The new window of [New Study] will appear. In this window, select [96 Wells Clear Plate] at the space of [Container Type] and click [Add Plate(s)...].
(3) Select your SDS file in the window with [File] tab. Then click [Add>>]. Your selected file name will appear in the space of [Plates To Be Added To Study]. Click [OK]. Click [OK] again.
(4) Click “small single green arrow” in the menu bar. The window of [Analysis Setting] will appear. Click [Detector] tab. Confirm that Automatic Ct and Automatic Baseline are selected. Click [Apply] and [OK].
(5) In the left lower part of the window, Ct number in each well will be demonstrated. Save this data.
(6) In SDS RQ manager, select [File], [Export] and [Amplification Data]. Save this file as, for example “Test”. You can find the icon of “Test.sdm-Amplification Data”. Drug this icon and release in an opened excel window.
(7) Using excel, you can normalize an mRNA expression level of “gene of your interest” by that of your “Endogenous Control” gene. In other words, the primary normalization, which is shown by “delta Ct”, can be done as follows: delta Ct = (Ct gene of your interest) – (Ct your endogenous control gene).
(8) Using excel, you can normalize “delta Ct in your tested samples” by “delta Ct in your control samples”. In other words, the secondary normalization, which is shown by “delta delta Ct”, can be done as follows: delta delta Ct = (delta Ct tested sample) – (the delta Ct control sample).
(9) Finally calculate the relative gene expression level by the following formula:
The relative gene expression level = 2-delta delta Ct.