ScreenTape® R6KWhat is the sensitivity of the ScreenTape R6K platform? ScreenTape can detect less than 1ng/µL starting RNA concentration. What is the Qualitative range of the ScreenTape R6K platform? 10ng/µL to 100ng/µL starting RNA concentration for the standard protocol Do I have to use ScreenTape RNA loading buffer? Yes – the RNA loading buffer contains a specific fluorescent dye and an RNA specific marker. What is the resolution of the platform? The RNA platform has the resolution necessary to analyse total RNA, cRNA and fragmented RNA from eukaryotic and prokaryotic samples. How much sample does the TapeStation load onto each lane of the ScreenTape R6K? 1µL What volume of sample is required for ScreenTape RNA analysis? The RNA sample should be diluted 1:1 with RNA loading buffer and the TapeStation will load from a minimum of 4µl in a PCR tube. It is therefore recommended that a minimum of 2µL total RNA sample is used at a concentration between 10 and 100ng/µl. What is the optimal RNA starting concentration for ScreenTape RNA analysis? For optimal ScreenTape software performance and ScreenTape Degradation Value (SDV) generation, an RNA starting concentration of between 20 and 100ng/µL is recommended. What plastic-ware should I use for sample preparation and TapeStation loading? It is recommended that RNase-free laboratory plastic-ware is used for sample preparation. Samples can be loaded into the TapeStation in 0.2ml individual PCR tubes or 0.2ml 8 tube PCR strips, additionally standard dimension 96 well PCR plates can be used for higher number sample batches. Do I need any additional specialist laboratory equipment to increase the efficiency of ScreenTape RNA analysis? There is no essential additional equipment required. Certain pieces of equipment will however make sample preparation easier and faster. What is the average sample preparation time for 16 RNA samples? 5 minutes which includes RNA denaturation How long can I keep my sample once it has been prepared to run on R6K? We recommend that the sample is run within 10 minutes of being prepared and that during this time it is stored on ice. What is the TapeStation sample processing time for 16 RNA samples? 12 minutes What are the storage conditions for the R6K ScreenTape and RNA loading buffer? ScreenTape R6K and RNA loading buffer should be stored at 4°C. What is the shelf life of R6K ScreenTape? Current shelf life product is 4 months at 4°C but new products under development will extend this shelf life further. What is the ScreenTape Degradation Value (SDV)? The ScreenTape Degradation Value (SDV) is an objective measurement of RNA degradation giving an RNA quality score on a scale of 0 to >200. Increasing SDV values indicate increasing RNA degradation. How do I interpret the ScreenTape Degradation Value (SDV)? SDV scores increase as RNA degradation increases and these are colour coded to assist with efficient QC processes. The default settings for colour coding are, Green (SDV 0 to 10), Amber (SDV 10 to 20) and Red (SDV>20). There is a user option in the GeneTools analysis software to select custom thresholds for both the green and amber colour coding selections. Ultimately SDV value thresholds should be set with the downstream application in mind, and will depend on the user’s experience of their experimental value within this context. How does the GeneTools software generate the ScreenTape Degradation Value (SDV)? The ScreenTape software determines the 18S peak height and the area of a region termed the phast zone between the 18S peak position and the small RNAs. These values are then used to calculate the ScreenTape Degradation Value (SDV) that is reported. How can I use ScreenTape to assess RNA integrity and characterization? The GeneTools software includes the combination of ScreenTape Degradation Value (SDV), peak profiles and ribosomal peak metrics, which provides you with enough data to objectively estimate RNA degradation and carry out rigorous RNA QC. Do I need to run a ScreenTape RNA ladder each time I run my samples? No it is not a requirement. The Lab901 platform has a saved RNA molecular weight marker that can provide an RNA profile for peak sizing. This facility avoids the use of a lane for the molecular weight marker and allows maximum use of all 16 lanes on the tape. If you do want to run a molecular weight marker, we recommend the use of the Norgen BioTek RNA ladder (Cat. nb. 15006) Can I save the image of a custom RNA ladder for use with subsequent samples? Yes Does the GeneTools software assign Molecular weight sizing according to the ladder that is run? Yes Is there a preferred RNA extraction method for samples run on the ScreenTape system? There is no preference as to how an RNA sample has been extracted as all samples can be analysed using the ScreenTape system. Separation profiles may differ depending on how the RNA has been purified, but results should be reproducible between samples purified following the same method. Why should the RNA samples be heat denatured before analysis? This is recommended to avoid secondary structures, which may affect the RNA run profiles. Heating the samples to 70°C for 2 min before loading onto the TapeStation will ensure complete denaturation and result in sharper RNA ribosomal peaks. There appears to be two bands evident in the ribosomal peak positions on the gel. What could be the cause of this? It is possible that the RNA samples were not sufficiently denatured during sample preparation. Please refer to your manual for optimal sample preparation conditions. |
