Set-up, programming and analysis RotorGene 3000/6000, Rotor-Gene Q

Transcription

1 Set-up, programming and analysis RotorGene 3000/6000, Rotor-Gene Q

2 How to start a Rotor-Gene run 1.Start the Rotor-Gene software. 2.Choose: Advanced/ Perform Last Run and press New.

3 How to start a Rotor-Gene run 1. Choose the Rotor you use (36 or 72 tubes) in the Rotor Selection field. 2. Tick Locking Ring Attached and press Next.

4 How to edit the set up of a run method 1. You can name the operator and write down some notes. 2. Choose 25ul as Reaction Volume and define your Sample Layout (1,2,3 ). 3. Press Next.

5 How to edit the set up of a run method 1. Tick the Green Channel. 2. Press Edit Gain and the small window Gain for Green appears. 3. Set the gain on Repeat the same for Yellow, Orange and Red. 5. Press Edit Profil.

6 How to edit the set up of a run method 1. Programme the FTD protocol (see table). 2. You can add the different steps/ temperatures/ times. Step temperature/time Reverse transcription (Hold) 50 C/ 15 min Hold 94 C/ 1 min Denaturation 94 C/ 8 sec Amplification 60 C/ 1 min Cycles 40

7 How to edit the set up of a run method 3. Programme the reverse transcription step. Step temperature/time Reverse transcription (Hold) 50 C/ 15 min Hold 94 C/ 1 min Denaturation 94 C/ 8 sec Amplification 60 C/ 1 min Cycles 40

8 How to edit the set up of a run method 4. Programme the second hold step. Step temperature/time Reverse transcription (Hold) 50 C/ 15 min Hold 94 C/ 1 min Denaturation 94 C/ 8 sec Amplification 60 C/ 1 min Cycles 40

9 How to edit the set up of a run method 5. Programme the cycling step (denaturation). Step temperature/time Reverse transcription (Hold) 50 C/ 15 min Hold 94 C/ 1 min Denaturation 94 C/ 8 sec Amplification 60 C/ 1 min Cycles 40

10 How to edit the set up of a run method 5. Programme the cycling step (amplification). Step temperature/time Reverse transcription (Hold) 50 C/ 15 min Hold 94 C/ 1 min Denaturation 94 C/ 8 sec Amplification 60 C/ 1 min Cycles 40

11 How to edit the set up of a run method 6. Within the cycling step choose the following four channels for acquisition of signals: - Green; - Orange; - Red; - Yellow; 7. Press OK.

12 How to insert and start a plate 1. After pressing OK, this window opens. 2. Open the cycler by shifting the lid backwards and insert the rotor. 3. After inserting your rotor containing your tubes (make sure that all positions are occupied) close the lid by sliding forward. 4. Choose Start Run and add the destination of your run file. 5.You have also the option to save a template for future runs.

13 How to edit the set up of a plate (defining samples) 1. After starting your run you choose Edit Sample. 2. Insert your Sample Name and Type and confirm with OK.

14 How to start analysing your plate 1. Press onto Analysis. 2. Double click one by one through the channels of interest (f.e. Yellow) and follow the next steps

15 How to start analysing your plate 1. Choose Linear Scale and, if necessary, modify the baseline with Slope Correct and Ignore first (e.g. 5). Using Outlier Removal is not recommended, as you could delete low positives.

16 How to start analysing your plate 1. In the picture above the Yellow channel was chosen. 2. To modify the threshold you need to click next to the threshold value (arrow). 3. In the table view you will find the Ct values. 4. You need to repeat these steps for all 4 channels.

17 FIRST: Check PC and NC. SECOND: Check patients 1. Now you are able to start with analysing your plate 2. Start with checking your positive, negative and internal control. 3. If your controls are valid ( kit manual) you can start to analyse your patient samples.

18 How to analyse a quantitative assay 1. At the sample defining step (see previous slides), press onto Edit sample. 2. The following window should pop-up. 3. Select the wells in which you put the quantification standards (QSx) and choose Standard from the drop-down list.

19 How to analyse a quantitative assay 4. Select the wells containing the same quantification standard QSx (red circle) and then add the corresponding concentration (red circle with arrow) 5. Please note that quantification standards need calibration ( for more info, consult our FAQ) If you proceed in that way, the same concentration will be applied to all the channels

20 How to analyse a quantitative assay 1. To be able to enter different concentrations for the different channels, press onto the button New at the bottom. 2. From here you can open different Pages. 3. You can move between and define the content of the different pages by pressing onto the arrows < and > that are next to the New button and give the Pages these different names (in box next to Name: ). 4. To speed up the process, you can just copy the information from the table that you defined in the first page and paste it into the other pages. 5. After that you can change the concentrations for the QSx in each Page for each channel.

21 How to analyse a quantitative assay 6. If you now go back to the Analysis window, the defined Pages are highlighted below each channel. Shown is an example for the FTD-1.1 ACE kit: FTD _64 - MANUAL - v4-2018_01 EN > Page 1: EBV-GREEN > Page 3: HCMV-YELLOW > Page 4: IC > Page 2: HAdV-RED 7. Select the Page that corresponds to the channel (green arrow appears in front) by double-clicking onto it. To remove a Page right-click onto the Page and select Remove Analysis.

22 How to analyse a quantitative assay 9. After having assigned the Pages, the regression analysis is done separately for each channel using the concentration values entered into the defined Pages. 10. If you have several PPmixes on the same plate, you could use the same principle to separate the different PPmixes. This would allow you to set an individual threshold for each PPmix.

23 If you have any further questions or suggestions, please contact us at or check out the FAQ section on our website: