Pathologists have long used formalin-fixed, paraffin-embedded (FFPE) tissue samples to examine morphology. With the advent of PCR amplification, these samples provide the potential for correlating morphology with genotype. Unfortunately, the process of tissue fixation results in cross-linking between proteins and DNA and introduction of inhibitor compounds. This reduces the efficiency of amplifying DNA, and as a consequence poor results generally have been observed with this sample type. DNA purification from formalin-fixed, paraffin-embedded tissues can be time consuming and tedious. The Maxwell® CSC System was developed to meet the needs of low- to moderate-throughput users by providing automated purification at a scale appropriate to their workload without considerable capital investment, training or maintenance. The prefilled cartridge design and mechanical plunger action of the Maxwell® CSC System make it ideal for simple, rapid purification of a wide variety of sample types. Here we demonstrate the utility of the Maxwell® CSC Instrument for automated purification of DNA from FFPE tissue samples. Using the prefilled cartridges of the Maxwell® CSC DNA FFPE Kit, DNA can be extracted from up to 16 samples at a time in approximately 40 minutes.
In this study, DNA was isolated from 23 single donor FFPE tissue samples using the Maxwell® CSC System. After isolation, DNA was quantitated using three methods: Absorbance at A260 using a NanoDrop® spectrophotometer, a specific fluorescent double-stranded DNA binding dye and fluorometer (Quantus™ Fluorometer/QuantiFluor® ONE dsDNA System), and a quantitative mulitiplexed qPCR system (Plexor® HY System). The DNA was diluted using the concentrations from both NanoDrop® and Quantus™ Fluorometer/QuantiFluor® systems, and those diluted samples were used in microsatellite instability analysis. We also compared GoTaq® MDx Hot Start Polymerase to AmpliTaq Gold® polymerase in the MSI Analysis System.
Figure 1. Workflow for the MSI analysis described in this study.
Materials and Methods
- 23X BioChain Institute Inc., Paraffin Tissue Sections, Male, 57 years old, Adenocarcinoma from colon, 5µm in thickness, one curl per sample tube
- Maxwell® CSC Instrument (Cat.# AS4000)
- Maxwell® CSC DNA FFPE Kit (Cat.# AS1350)
- Quantus™ Fluorometer (Cat.# E6150)
- QuantiFluor® ONE dsDNA System (Cat.# E4871)
- K652 Genomic DNA (Cat.# E4931)
- NanoDrop® 1000 spectrophotometer
- Plexor® HY System (Cat.# DC1001)
- MSI Analysis System, Version 1.2 (Cat.# MD1641)
- GoTaq® MDx Hot Start Polymerase (Cat.# D6001)
- AmpliTaq Gold® DNA polymerase (Applied Biosystems)
- ABI 3500xL Genetic Analyzer
- GeneMapper® 5.0 software (Applied Biosystems)
- PowerPlex® Matrix Standards, 3100/3130 (Cat.# DG4650)
- Hi-Di™ formamide (Applied Biosystems, 4311320)
DNA was extracted from 23 colon tissue FFPE samples using the Maxwell® CSC DNA FFPE Kit according the manufacturer's instructions (Technical Manual #TM395).
To measure DNA using the NanoDrop® 1000 spectrophotometer, 2µl of purified DNA from each sample was used and measured according to manufacturer's instructions.
To measure DNA using the dye method, 2µl of each eluate was added to 198µl of QuantiFluor® ONE reagent, mixed briefly and the concentration was measured using the Quantus™ Fluorometer. Manufacturer's instructions in Technical Manual #TM405 were followed for calibrating the instrument and measuring DNA concentration using the Quantus™ Fluorometer. K562 Genomic DNA was used to calibrate the instrument.
To measure DNA using the qPCR method, the manufacturer instructions for setup and data analysis using the Plexor® HY System were followed (Technical Manual #TM293). DNA eluates were diluted 1:10 in TE buffer, and a volume of 2µl was added to the amplification mixture.
Amplification using MSI Analysis System, Version 1.2
The DNA eluates were diluted twice to 1ng/µl in water based on concentrations from NanoDrop® and Quantus™ Fluorometer/QuantiFluor® ONE dsDNA System analyses.
Amplification setup was performed as described in the MSI Analysis System Version 1.2 Technical Manual #TM255 using either GoTaq® MDx Hot Start Polymerase or AmpliTaq Gold® DNA polymerase. A total volume of 2µl of each diluted DNA sample was added to the reaction mixture. Amplification thermal cycling was performed as outlined in the MSI Analysis System Version 1.2 Technical Manual #TM255 on a GeneAmp® PCR System 9700 Thermal Cycler.
Detection of Amplified Fragments using the ABI 3500xl Genetic Analyzer
After amplification, 1µl of each amplification sample was added to a mixture of 0.5µl ILS 600 and 9.5µl Hi-Di™ formamide. K562 Genomic DNA from the MSI Analysis System was used as a positive control and water was used as a negative control. After a brief centrifugation, the samples were denatured at 95°C for 3 minutes then immediately chilled on ice for 3 minutes. The instrument was prepared and fragments detected as described. However in this experiment we used POP-4™ polymer instead of POP-7™ polymer. Spectral calibration was performed using the PowerPlex® Matrix Standards 3100/3030. The instrument was installed with a 36cm capillary array.
MSI Data Analysis
Data were analyzed using GeneMapper® Software, version 5.0, with panel and bin files downloaded from www.promega.com/resources/tools/msi-panels-and-bins-for-genemapper-software/. The data were processed by selecting the maximum peak height within a locus and averaging those values for all samples.
DNA Isolation and Quantitation
DNA was isolated from 23 human colon FFPE samples using the Maxwell® CSC DNA FFPE Kit. Eluates were collected and concentrations determined using three methods: NanoDrop® 1000 spectrophotometer, Quantus™ Fluorometer/QuantiFluor® ONE dsDNA System, and the Plexor® HY System (Figures 2 and 3). The NanoDrop™ 1000 results gave the hightest concentration readings of the three methods. This can be explained in part because this method measures all of the nucleic acid in the sample, including the RNA. The Quantus™ Fluorometer/QuantiFluor® ONE dsDNA System preferentially binds dsDNA and is therefore a more accurate measure of the DNA present in a sample. The Plexor® HY System provided concentration readings intermediate between the other methods. The Plexor® HY method measures the total amplifiable DNA present in a sample, both single- and double-stranded (99 base pair target size).
The DNA eluates were diluted to 1ng/µl based on both the Quantus™ Fluorometer/QuantiFluor® System concentrations and the NanoDrop® 1000 concentrations. A total volume of 2µl of each dilution was used in 10µl MSI amplification reactions using GoTaq® MDx Hot Start Polymerase with MSI primers. After amplification, the reactions were analyzed using a 3500xL ABI genetic analyzer (Figure 4). All seven loci were detected from all amplification reactions and could be completely overlaid. The samples diluted based on the Quantus™ Fluorometer/QuantiFluor® results consistently gave higher RLUs compared to samples diluted based on the NanoDrop® 1000-determined concentrations (Figure 5). We also compared amplification using GoTaq® MDx Hot Start Polymerase to amplification using AmpliTaq Gold® DNA polymerase (Figure 6). We found very similar peak heights between the two samples, showing similar results between the two polymerases (Figure 7).
Figure 6. Sample electropherogram of MSI analysis of samples amplified with either AmpliTaq Gold® (Panel A) or GoTaq® MDx (Panel B) Hot Start Polymerases.
DNA extracted from 23 FFPE samples using the Maxwell® CSC DNA FFPE Kit was compatible with downstream PCR-based analyses such as the Plexor® HY and MSI Analysis Systems, and in MSI analysis all seven loci were detected from the samples analyzed. When quantitating DNA obtained from the FFPE samples, higher values were obtained from the NanoDrop® spectrophotometer, presumably because it measures all nucleic acid present in the sample, whereas the Plexor® HY System detects only amplifiable DNA (either single- or double-stranded) and the Quantus™ Fluorometer/QuantiFluor® Dye method preferentially detects and measures double-stranded DNA. Samples that were diluted based on concentrations determined using the Quantus™ Fluorometer/QuantiFluor® Dye methods resulted in higher RFU values in the MSI analysis than those diluted based on concentrations determined using the NanoDrop® 1000. This indicates that the Quantus™ Fluorometer/QuantiFluor® Dye method is superior for quantitating low abundant DNA samples before MSI analysis. Additionally, in our hands, no difference was observed in MSI analysis performed using GoTaq® MDx Hot Start Polymerase or AmpliTaq Gold® polymerase.